La realizzazione di questo volume
è stata possibile per gentile concessione
di LI-COR Bioscences
Bi o s c i e n c e s
IMAGING INFRAROSSO
L'analisi quantitativa dell'espressione proteica
INDICE
1. L’uso dell’Infrarosso per una maggiore accuratezza
1
nell'analisi quantitativa dei Western Blot
I vantaggi della fluorescenza nel campo
della Vicino Infrarosso (NIR, Near InfraRed)
Uso della tecnologia ad infrarossi anche in altre applicazioni
Referenze
1. Applicazioni
2. Protocolli Applicativi
7
9
Western Blot Analysis
In-Cell Western Assay
4. Pubblicazioni
5. Prodotti per Imaging Infrarosso
23
51
Fluorofori compatibili con Odyssey
Guida ai prodotti LI-COR
6. Contattare M-Medical
63
Indice
L’INFRAROSSO NELL’ANALISI QUANTITATIVA
IMAGING INFRAROSSO
L'analisi quantitativa dell'espressione proteica
L’USO DELL’INFRAROSSO PER UNA MAGGIORE
ACCURATEZZA NELL'ANALISI QUANTITATIVA
DEI WESTERN BLOT
I sistemi di imaging per Western Blot sono spesso descritti come metodi quantitativi. Questa definizione è vera solamente se lo strumento acquisisce l’immagine del blot rispettando precisi requisiti necessari ad un processo quantitativo.
I principali requisiti che si rendono necessari a definire un processo di acquisizione di immagine come processo quantitativo sono:
Proporzionalità diretta tra segnale generato e quantità di campione
presente sul blot
Linearità del rapporto proporzionale
In altre parole un sistema si può definire quantitativo quando, al raddoppiare
della quantità di un campione su un blot, si osserva un aumento proporzionale
del segnale.
Storicamente il metodo più diffuso per l’identificazione di presenza di un antigene specifico su un blot è la chemiluminescenza, associata sia alle pellicole
fotografiche che ai sistemi basati su camere digitali raffreddate (CCD cameras).
La responsabilità della grande diffusione di questo metodo è da attribuire alla
maggiore sensibilità che il metodo chimico di luminescenza ha saputo garantire.
La chemiluminescenza consiste nella generazione di fotoni di luce per reazione
chimica di un substrato, catalizzata da un enzima (coniugato all’anticorpo se
facciamo riferimento al Western Blotting), per la determinazione di questo segnale si espone una lastra fotografica o la CCD camera (che elabora l'immagine
con un software) al blotting sul quale la reazione viene fatta avvenire.
La produzione di questa luce per reazione chimica catalizzata è dinamica, ovvero cambia in funzione del tempo. I campioni con quantità di proteina più abbondante producono maggiore luce in un tempo breve, i campioni con minori
quantità di proteina possono produrre quantità di luce paragonabili ai campioni
più abbondanti, ma in un tempo più lungo. Le immagini devono essere quindi
catturate ad un intervallo di tempo ottimizzato dall’inizio della reazione chimica.
Questa dipendenza della quantità di segnale prodotto in funzione del tempo
compromette significativamente l’accuratezza del saggio quantitativo.
Al contrario della chemiluminescenza, l’emissione di fluorescenza è statica;
quando un fluoroforo viene eccitato, l’emissione di fluorescenza ad una lunghezza d’onda superiore rispetto l’eccitazione è costante.
1
Questo permette di considerare la rivelazione di fluorescenza un metodo più
accurato e preciso per l’analisi quantitativa di proteine. La fluorescenza può essere sfruttata coniugando molecole fluorescenti agli anticorpi usati nel Western
Blotting.
In passato questo approccio non ha avuto successo per la scarsa sensibilità garantita dall’uso della fluorescenza dello spettro delle radiazioni elettromagnetiche visibili. La tecnologia ad Infrarossi progettata e sviluppata da LI-COR
garantisce elevati livelli di sensibilità, conservando i vantaggi quantitativi offerti
dalla Fluorescenza.
I vantaggi della fluorescenza nel campo della Vicino
Infrarosso (NIR, Near InfraRed)
Alta sensibilita`
Mean Membrane Signal
La fluorescenza nel campo del vicino infrarosso garantisce una sensibilità paragonabile o addirittura superiore rispetto alla chemiluminescenza, tenendo conto
che la sensibilità della stessa chemiluminescenza dipende dal tipo di substrato
e dal metodo di identificazione usato (film o CCD camera). Alle lunghezze d’onda superiori a 680 nm le superfici delle membrane e le biomolecole esprimono
un’autofluorescenza nettamente inferiore rispetto alla fluorescenza emessa nel
campo del visibile, generando un background molto basso, che si traduce in
una sensibilità molto alta. Le membrane di nitrocellulosa e PDVF scansionate nel
vicino infrarosso hanno una autofluorescenza enormemente inferiore rispetto all’autofluorescenza misurata nello spettro visibile, come rappresentato in Figura 1.
Ne risulta una forte riduzione del background associato alla membrana. La riduzione del background garantito
Infrared vs. Visible Wavelength Membrane Fluorescence
dall’analisi della fluorescenza
ad infrarosso rende questo tipo
3000000
di approccio paragonabile alla
PVDF
2500000
chemiluminescenza in termini
Nitrocellulose
di sensibilità.
2000000
Fig,. 1
Le membrane di PVDF sono state
scansionate su Odyssey Infrared
Imagin System (LI-COR Bioscience)
con intersità = 5 per entrambe le
lunghezze d’onda, 700 e 800 nm.
1500000
1000000
500000
Le stesse sono state scansionate
a 532 e 635 nm su GenePix
4100° (Molecular Devices).
0
800nm
700nm
532nm
635nm
Wavelength
2
L’USO DELL’INFRAROSSO PER UNA MAGGIORE ACCURATEZZA
NELL'ANALISI QUANTITATIVA DEI WESTERN BLOT
Ampio spettro di linearita`
L’uso della fluorescenza permette di ottenere un intervallo di linearità maggiore
in un Western Blot rispetto a quello che la chemiluminescenza può dare (1).
Questo importante aspetto è ben rappresentato nella Figura 2: con la chemiluminescenza l’uso di un tempo di esposizione (10 minuti) ottimizzato per raccogliere i dati relativi alle bande in cui la quantità di proteina è molto bassa (1
pg), portano ad avere delle bande non analizzabili per quantità di proteine più
abbondanti (1000 pg) (Figura 2E). Allo stesso tempo usando tempi di esposizione (30 secondi) ottimizzati per analizzare le proteine più abbondanti, non si
ottengono dati per le proteine meno abbondanti (Figura 2B).
Quando si analizzano proteine non bene caratterizzate è possibile che la presenza e la quantità di una determinata proteina possa non essere identificata affatto, solamente a causa dell’uso di un tempo di esposizione non ottimizzato.
Con la tecnologia ad Infrarossi, grazie alla emissione statica di fluorescenza e
all’ampio intervallo di linearità, è possibile analizzare con una singola scansione
concentrazioni molto diverse di proteine presenti sullo stesso blot.
Fig. 2
Confronto tra un acquisizione di fluorescenza infrarossa
e chemiluminescenza di transferrina alle concentrazioni
1000 pg, 32 pg e 1 pg, su membrana di nitrocellulosa.
(A) scansione con Odyssey a 800 nm con intensità = 4.
(B) chemiluminescenza su film dopo
0 secondi di esposizione.
(C) chemiluminescenza su film dopo
1 minuto di esposizione.
(D) chemiluminescenza su film dopo
10 secondi di esposizione.
3
Identificazione simultanea di due proteine
I meccanismi di trasduzione del segnale intracellulare sono spesso regolati
da degli eventi di fosforilazione proteica. Proteine come recettori tirosin-chinasi
possono essere fosforilate in seguito al legame con il loro ligando naturale, innescando così meccanismi plurimi di attivazione metabolica e biochimica. Grazie
all’uso dello strumento ODYSSEY, alle sue due sorgenti di eccitazione diverse
ed ai due sistemi di rilevazione distinti, rispettivamente a 700 e 800 nm, è possibile misurare la quantità di proteina presente in un campione e contemporaneamente la quantità relativa della stessa proteina nella forma fosforilata, nonostante
le due abbiano un peso molecolare pressoché identico (Figura 3).
Fig. 3
Anti-EGFR e Anti fosfo-EGRF
in cellule A431. La figura
rappresenta diluizioni seriali
1:2 di lisati cellulari di A431.
Nell’immagine (A) è possibile
visualizzare lo shift elettroforetico
causato della fosforilazione.
Questo metodo di quantificazione simultanea aumenta sensibilmente la
accuratezza delle analisi
quantitative su immunoblotting; un canale di analisi viene usato per misurare
la quantità di una determinata proteina di interesse, e
l’altro canale viene utilizzato
per normalizzare la quantità di proteine totali presenti per ogni campione.
La tecnologia a due canali nel campo del vicino infrarosso rende possibile la
quantificazione accurata di proteine presenti in quantità molto basse (3), incrementando la precisione grazie alla possibilità di normalizzare sempre i propri
dati, caratteristiche molto complicate da ottenere con altri metodi; la chemiluminescenza ha una alta sensibilità, ma non consente di avere linearità e proporzionalità dei dati quantitativi ed inoltre non permette di normalizzare, ad esempio,
la quantità di proteina nella forma fosforilata sulla quantità di proteina totale, per
assenza di un secondo canale di acquisizione. La impossibilità di effettuare tali
normalizzazioni limita l’accuratezza nella quantificazione, soprattutto per quantità molto basse di proteina.
4
L’USO DELL’INFRAROSSO PER UNA MAGGIORE ACCURATEZZA
NELL'ANALISI QUANTITATIVA DEI WESTERN BLOT
Semplificazione delle procedure
Un ulteriore vantaggio della tecnologia ad infrarossi è la semplificazione delle procedure di Western Blot. Substrati, pellicole e camera oscura non sono più necessari. La fluorescenza infrarossa è stabile per mesi e le membrane possono essere
scannerizzate diverse volte e dopo diverso tempo senza perdita di segnale.
Uso della tecnologia ad infrarossi anche in altre applicazioni
Sfruttando le caratteristiche appena elencate, questo tipo di tecnologia si estende ad un ampio spettro di applicazioni che va oltre il Western Blotting.
In-Cell Western Assay
Questo metodo sviluppato da LI-COR consiste in un saggio di immunocitofluorecenza per quantificare le proteine direttamente in cellule in coltura. Determinare la quantità di proteine direttamente nel loro contesto cellulare incrementa
ulteriormente la precisione della quantificazione. Le proteine vengono determinate nelle cellule fissate direttamente nelle micropiastre nelle quali vengono fatte
crescere, incrementando il numero di campioni che possono essere analizzati
contemporaneamente rispetto ad un Western Blot, eliminando delle fasi critiche
che possono alterare l’accuratezza della quantificazione, tra cui la lisi cellulare,
la preparazione delle proteine, l’elettroforesi ed il trasferimento delle proteine
sulle membrane.
In-Cell Western Assay è già ampliamente diffuso ed è stato impiegato per diversi lavori che vanno dallo studio dei meccanismi di trasduzione del segnale
intracellulare allo studio dei recettori a sette domini transmembrana accoppiati
alle proteine G, dalla neurologia alla oncologia (3,4,5). Una variazione al metodo
In-Cell Western è stata applicata allo studio dell’internalizzazione e riciclaggio
del recettore dei cannabinoidi CB1, appartenente alla superfamiglia dei recettori
a sette domini transmembrana accoppiati alle proteine G(6).
In Vivo Imaging
L’applicazione della tecnologia ad Infrarossi ha trovato spazio anche in studi di
“Imaging In Vivo” applicato a piccoli animali. La bassa fluorescenza dei tessuti
a 800 nm permette di usare sonde marcate con fluorofori emittenti a questa
lunghezza d’onda, per ottenere immagini di organi o tumori (7). L’”Imaging In
Vivo” è importante per ogni ricerca che comprenda l’uso di modelli animali per
lo studio di patologie, come ad esempio la malattia di Alzheimer (8).
La tecnologia ad Infrarossi è stata utilizzata con ampio successo anche per altre
applicazioni quali lo studio morfologico di sezioni di tessuto (9), Protein Arrays
(10), ed EMSA assay (11).
5
Referenze
1. Schultz.Geschwender, A., Zhang, Y., Holt, t., McDermitt, D., And Olive
2.
3.
4.
5.
6.
7.
8.
9.
D.M. (2004). Quantitative, two-colours western blot detection with
infrared fluorescence. http://www.licor.com/bio/PDF/IRquant.pdf
Picariello L, Sala SC, Martineti V, Gozzini A, Aragona P, Tognarini I, Paglierani
M, Nesi G, Brandi ML, Tonelli F. (2006).
A comparison of methods for the analysis of low abundance proteins in
desmoid tumor cells.
Anal. Biochem. 2006 Jul 15;354(2):205-12.
Chen H, Kovar J, Sissons S, Cox K, Matter W, Chadwell F, Luan
P, Vlahos CJ, Schutz-Geschwender A, Olive DM. A cell-based
immunocytochemical assay for monitoring kinase signaling pathways
and drug efficacy. Anal Biochem. 2005 Mar 1;338(1):136-42.
Wong SK. A 384-well cell-based phospho-ERK assay for dopamine D2 and
D3 receptors.
Anal Biochem. 2004 Oct 15;333(2):265-72.
Dickey CA, Eriksen J, Kamal A, Burrows F, Kasibhatla S,
Eckman CB, Hutton M, Petrucelli L. Development of a high
throughput drug screening assay for the detection of changes
in tau levels -- proof of concept with HSP90 inhibitors.
Miller, J. Tracking G protein-coupled receptor trafficking using Odyssey
imaging. http://www.licor.com/bio/PDF/Miller _ GPCR.pdf
Houston, J.P., Ke, S., Wang,W., Li, C., Sevick-Muraca, E.M.. Quality analysis
of near-infrared fluorescence and conventional gamma images acquire using
dual labelled tumor targeting probe. J.Biomed.Optics. 10:054010-1-11.
Skoch J, Bacskai B. The LI-COR Odyssey as a near-infrared imaging platform
for animal models of Alzheimer’s Disease.
http://www.licor.com/bio/PDF/MassGen.pdf
Hawes JJ, Brunzell DH, Wynick D, Zachariou V, Picciotto MR.
GalR1, but not GalR2 or GalR3, levels are regulated by galanin
signaling in the locus coeruleus through a cyclic AMP-dependent
mechanism. J Neurochem. 2005 Jun;93(5):1168-76.
10. Yeretssian G, Lecocq M, Lebon G, Hurst HC, Sakanyan V.
Competition on nitrocellulose-immobilized antibody arrays:
from bacterial protein binding assay to protein profiling in breast
cancer cells. Mol Cell Proteomics. 2005 May;4(5):605-17.
11. Geddie ML, O’Loughlin TL, Woods KK, Matsumura I. Rational design of p53,
an intrinsically unstructured protein, for the fabrication of novel molecular
sensors. J Biol Chem. 2005 Oct 21;280(42):35641-6. Epub 2005 Aug 23.
6
L’USO DELL’INFRAROSSO PER UNA MAGGIORE ACCURATEZZA
NELL'ANALISI QUANTITATIVA DEI WESTERN BLOT
APPLICAZIONI
IMAGING INFRAROSSO
L'analisi quantitativa dell'espressione proteica
APPLICAZIONI
Analisi dell'espressione proteica nel Vicino Infrarosso.
Elenco delle applicazioni:
Western Blotting quantitativo
In-Cell Western
Protein Array
FLISA (Fluorescence Linked Immuno-Adsorbant Assay)
Analisi Morfologica di sezioni di tessuto
Imaging in piccoli animali vivi
Odyssey può essere sfruttato anche per l’analisi di acidi nucleici attraverso diverse metodologie ed approcci analitici.
In particolare, Odyssey può essere utilizzato per:
Raccogliere ed analizzare immagini di Staining degli Acidi
Nucleici Totali (analisi di restrizione del DNA ed analisi di
prodotti di PCR)
Northern e Southern Blotting
EMSA
Analisi di cDNA AFLP con recupero delle bande di cDNA
7
PROTOCOLLI APPLICATIVI
IMAGING INFRAROSSO
L'analisi quantitativa dell'espressione proteica
PROTOCOLLI APPLICATIVI
Odyssey Infrared Imaging System è stato sviluppato in principio da LI-COR
per l’analisi quantitiva di proteine in Western Blotting. Abbiamo già descritto in
precedenza come Odyssey possa essere impiegato per l’identificazione e la
quantizzazione di proteine anche in altri contesti, come per esempio l’analisi in
colture cellulari o l’analisi in piccoli animali vivi.
Questa sessione della Guida illustra i protocolli applicativi ottimizzati da LI-COR
per alcune di queste applicazioni. Ulteriori informazioni sui protocolli applicativi
e sulle altre applicazioni possono essere consultate alla pagina:
http://biosupport.licor.com
9
Protocollo Western Blot Analysis
Reagenti necessari
Filtri di nitrocellulose o PVDF
“Odyssey Blocking Buffer”
Anticorpi primari
Anticorpi secondari marcati con IR(dyes)
Tween20
Tampone di lavaggio PBS
Acqua bidistillata
Metanolo per PVDF
SDS
Altri tamponi di bloccaggio se necessari
Lista dei Fluorofori IR(dyes) piu` appropriati per l uso
su Odyssey
FLUOROFORO
SENSIBILITÀ
CANALE ODYSSEY
IRDye 800CW
+++
800
IRDye 800
+++
800
IRDye 680
+++
700
IRDye 700DX
++
700
AlexaFluor 680
+++
700
AlexaFluor 700
++
700
AlexaFluor 647
+
700
++
700
+
700
Cy5.5
Cy5
Metodo
Sia le membrane in nitrocellulosa che le membrane in PVDF possono essere
utilizzate per il blotting di proteine, ma la nitrocellulosa è maggiormente raccomandata se si richiedono performance di sensibilità maggiori, grazie alla sua
minore autofluorescenza. La procedura di blotting per Western su Odyssey è
la procedura standard e le membrane devono essere maneggiate solo ai bordi
con pinzette pulite.
11
Prima del trasferimento delle proteine su membrana procedere con i seguenti
passaggi:
1. Bagnare la membrana per diversi minuti in PBS. Se usate PVDF pre-
asciugato, lavatelo ancora con 100% metanolo e sciacquatelo due volte
con acqua bidistillata prima di procedere all’equilibratura in PBS.
NOTA: L’inchiostro di molti tipi di penna può fluorescere su Odyssey. Usare una
matita o la penna accessoria di Odyssey per evitare questo inconveniente.
2. Effettuare il blocking dei siti aspecifici di adsorbimento della membrana
mediante incubazione in blocking buffer per 1 ora. La membrana deve
essere coperta interamente dal buffer.
NOTE: Le membrane possono essere bloccate overnight a 4°C.
Non usare Tween-20 durante il blocking, questo potrebbe provocare un alto
background se usato in questa fase.
Odyssey Blocking Buffer è stato ottimizzato per avere le migliori performance di
blocking. Latte in polvere o caseina sciolti in PBS possono essere usati sia per il
blocking che per le incubazioni degli anticorpi, facendo attenzione però ad usare il
latte su PVDF perché potrebbe causare un alto background. Nel caso si preferisca
usare la caseina, soluzioni al 0,1 o 0,2 % in PBS sono preferite. Non è richiesta
caseina a purezza Hammersten.
Blocking a base di latte possono interferire con la detection del segnale se si usano
anticorpi anti-goat.
Odyssey blocking buffer può essere diluito 1:1 in PBS.
Odyssey blocking buffer può essere recuperato e usato diverse volte.
Soluzioni di blocking contenenti BSA possono essere impiegate, ma in
alcuni casi possono dare livelli di background maggiori. Blocking buffer
contenenti BSA sono non generalmente consigliati, ma possono essere
usati quando l’anticorpo primario richiede l’uso di BSA come bloccante.
3. Diluire l’anticorpo primario in Odyssey Blocking Buffer. La diluizione otti-
male dipende dal tipo di anticorpo primario e deve essere stabilita empiricamente. Una diluizione di partenza suggerita è compresa nel range
compreso tra 1:1.000 e 1:5.000. Per ridurre il background aggiungere
0,1-0,2 % di Tween-20 nell’anticorpo primario diluito. La concentrazione
ottimale di Tween-20 dipende dall’anticorpo.
NOTA: L’analisi di due colori contemporaneamente richiede che
gli anticorpi primari siano di due specie diverse.
4. Incubare la membrana (dopo il trasferimento delle proteine dal gel) con
l’anticorpo primario per almeno 60 minuti a temperatura ambiente e
sotto costante e blanda agitazione (il tempo di incubazione ottimale
dipende dal tipo di anticorpo primario). Usare abbastanza soluzione di
anticorpo primario per ricoprire completamente la membrana.
12
PROTOCOLLI APPLICATIVI
5. Lavare la membrana 4 volte per 5 minuti ciascuno a temperatura am6.
biente in PBS +0,1% Tween-20, sempre sotto costante agitazione,
usando una quantità generosa di tampone.
Diluire l’anticorpo secondario marcato con il fluoroforo in Odyssey
Blocking Buffer. Evitare lunghe esposizioni della provetta contenente gli
anticorpi alla luce diretta. La diluizione raccomandata è di 1:15.000 (o
un range tra 1:5.000 e 1:25.000). Aggiungere Tween-20 alla diluizione
dell’anticorpo secondario se si è fatto per l’anticorpo primario.
NOTE: Per l’identificazione di piccole quantità di proteine, provare ad aumentare la
concentrazione di secondario a 1:5.000 – 1:10.000.
La diluizione dell’anticorpo secondario può essere recuperata dopo l’incubazione
e riutilizzata per altri blotting. Se si desidera riutilizzare la diluizione di anticorpo,
conservarla al buio a 4°C. Per avere le migliori performance in termini di sensibilità,
usare sempre diluizioni fresche appena preparate.
L’aggiunta di 0,01% - 0,02% di SDS alla diluizione di anticorpo secondario può
ridurre sensibilmente il background della membrana, in particolare di PVDF. Tuttavia
NON aggiungere SDS durante il Blocking o alla diluizione di anticorpo primario.
6. Incubare il blotting nella diluizione di anticorpo secondario per 30-60
7.
8.
minuti a temperatura ambiente sotto blanda agitazione. Proteggere dalla luce.
Lavare la membrana 4 volte per 5 minuti ciascuno a temperatura ambiente in PBS +0,1% Tween-20, sempre sotto costante agitazione,
usando una quantità generosa di tampone e proteggendo dalla luce.
Riprendere le membrane con PBS per rimuovere completamente
Tween-20. Le membrane sono così pronte per essere analizzate con
Odyssey.
NOTE: Proteggere la membrana dalla luce fino al momento della scansione.
Mantenere la membrana umida se si ha in programma di effettuare uno stripping.
È possibile far asciugare le membrane prima della lettura con Odyssey. La forza del
segnale può aumentare sulla membrana asciutta.
Il segnale di fluorescenza sulla membrana dura per diversi mesi ed oltre se
conservata al buio. Le membrane possono essere conservate asciutte a
temperatura ambiente o in PBS a 4°C.
Se il segnale ottenuto dalla scansione è troppo alto o troppo debole, riscannerizzare
la membrana con intensità di scansione più basse o più alte rispettivamente.
Marcatori di peso molecolare
Marcatori di peso molecolare colorati in blu sono visibili su Odyssey a 700 nm.
Caricare 1/3 o 1/5 della quantità normalmente impiegata nei western blot. Quantità troppo alte possono dare segnali così forti da interferire con i segnali dei propri campioni. Se vengono utilizzati marker multicolore, alcune bande potrebbero
non essere visualizzabili su Odyssey.
13
Consigli per l'ottimizzazione
Non esiste una soluzione di blocking universale ottimale per ogni
coppia antigene-anticorpo. Alcuni anticorpi primari possono dare
segnali molto deboli con alte aspecificità o migliorare le loro performance a seconda del sistema di blocking utilizzato. Nel caso si dovesse incontrare difficoltà nell’identificazione specifica di una banda,
il cambiamento della soluzione di blocking può aiutare sensibilmente
le performance.
Se un anticorpo primario funziona molto bene in chemiluminescenza
con un sistema di blocking, usare questo sistema anche per le analisi su Odyssey.
Per evitare la presenza di picchi di background sul blotting, usare
grandi quantità di acqua bidistillata per lavare ogni vaschetta di
incubazione. Non usare vaschette già impiegate per Coomassie
Staining.
Prima di effettuare una scansione, e al termine di ogni scansione,
lavare accuratamente la superficie di scansione di Odyssey.
Non avvolgere le membrane nella plastica per le scansioni.
Linee guida per la detection contemporanea di due colori
Odyssey permette di analizzare due diversi antigeni contemporaneamente sullo
stesso blotting usando due anticorpi secondari marcati con due diversi fluorofori (700 e 800 nm). Questo tipo di analisi richiede una scelta molto accurata
degli anticorpi primari e secondari. A seguire vengono elencati alcuni pratici
consigli per ottimizzare questo tipo di analisi:
I due anticorpi primari devono essere stati prodotti in due specie diverse, quindi possono essere riconosciuti da due anticorpi secondari diversi, marcato ciascuno con un fluoroforo diverso, rispettivamente
per 700 e 800 nm.
Prima di eseguire un esperimento di analisi dei due antigeni in contemporanea, eseguire l’analisi di ciascun antigene separatamente,
così da ottimizzare le condizioni d’uso degli anticorpi e ridurre al massimo gli eventi di cross-reattività tra gli anticorpi primari e secondari.
Per ridurre al massimo la cross-reattività evitare di usare secondari
anti-mouse e anti-rat contemporaneamente.
Se possibile, usare anticorpi secondari prodotti nella stessa specie,
in modo da evitare che si riconoscano l’uno con l’altro.
14
PROTOCOLLI APPLICATIVI
Ottimizzazione di un Western Blot
Quando si mette a punto un protocollo di Western Blot usando una nuova coppia di anticorpi primario e secondario, il punto più critico è quello dell’ottimizzazione delle concentrazioni degli anticorpi, in modo da avere i migliori risultati in
termini di specificità e di sensibilità.
I parametri critici sui quali è possibile intervenire sono:
Concentrazione dell’anticorpo primario
La concentrazione ottimale dipende molto dal tipo di anticorpo primario e dalla quantità di antigene che si identificherà nel campione.
Le quantità suggerite sono 1:500, 1:1.500, 1:5.000 e 1:10.000, oppure
può convenire partire dalla stessa concentrazione usata con successo in chemiluminescenza.
Concentrazione dell’anticorpo secondario
Anche la concentrazione di anticorpo secondario può essere determinata in modo empirico. I valori di concentrazione consigliati sono
1:5.000, 1:10.000 e 1:20.000. In ogni caso la quantità di anticorpo
secondario da utilizzare dipende molto dalla quantità di antigene da
rilevare. Maggiore è la quantità di antigene presente minore sarà la
quantità di anticorpo secondario da utilizzare.
Concentrazione dei detergenti nelle diluizioni degli anticorpi
L’aggiunta di detergenti alle diluizioni di anticorpi permette di migliorare sensibilmente i valori di background sul blot. La concentrazione
ottimale del detergente dipende da anticorpi, tipo di membrana e
bloccante usati. È importante ricordare che il legame tra antigene e
anticorpo primario non è particolarmente forte e un uso sbagliato dei
detergenti potrebbe impedire il corretto riconoscimento anticorpale e
ridurre la sensibilità del saggio.
Tween-20
Non usare questo detergente durante le fasi di blocking, potrebbe
provocare un alto background.
Aggiungere Tween-20 alle diluizioni di anticorpi primari e secondari.
Usare concentrazioni finali di 0,1%- 0,.2% con membrane di nitrocellulosa e 0,1% su PVDF.
Anche le soluzioni di lavaggio possono contenere lo 0,1% di Tween-20.
SDS
Aggiungere 0,01–0,02 % di SDS alla diluizione dell’anticorpo secondario può aiutare sensibilmente a ridurre il background e ridurre
15
l’aspecificità dell’anticorpo. SDS deve essere usato sempre a basse
concentrazioni, per il suo carattere tensioattivo può negativamente
intervenire sul riconoscimento antigene-anticorpo.
Non aggiungere mai SDS nelle fasi di blocking o alla diluizione dell’anticorpo primario.
Non aggiungere SDS alle soluzioni di lavaggio.
SDS aiuta a ridurre il background occasionalmente dovuto ai blocking
con BSA.
Protocollo Acquisizione ed analisi di gel di proteine colorati
con Blue-Coomassie
Odyssey può essere usato anche per gel documentazione ed analisi di gel
di proteine colorati con Blue-Coomassie. I coloranti a base di Blue-Coomassie, sia a base acquosa che a base alcolica, emettono fluorescenza chiara a
700 nm e fluorescenza più labile a 800 nm. La visualizzazione effettuata con
Odyssey è centinaia di volte più sensibile della visualizzazione ad occhio nudo,
proprio per l’uso delle lunghezze d’onda infrarosse e non visibili percepite dall’occhio umano. I coloranti a base acquosa permetto generalmente di ottenere
livelli di sensibilità migliori, performance ulteriormente migliorabili con procedure
di “destaining” in acqua overnight.
Protocollo In-Cell Western Assay
Nella descrizione del metodo verrà usato come esempio l’espressione di fosfoEGFR e fosfo-ERK in cellule A432, in risposta a stimolo con Epidermal Growth
Factor.
Reagenti Odyssey necessari
Anticorpo secondario Anti-Mouse marcato con IRDye™ 800CW
Anticorpo secondario Anti-Rabbit marcato con IRDye™ 680CW
Odyssey blocking buffer
Altri reagenti
1X PBS
Reagenti per colture cellulari (siero, DMEM, tripsina e PBS)
20% Tween®-20
Epidermal Growth Factor
37% formaldeide
16
PROTOCOLLI APPLICATIVI
10% Triton®X-100
Micropiastre 96 pozzetti per colture cellulari
Anticorpi primari per gli antigeni in esame
Semina, stimolazione e determinazione della risposta
su cellule A431
1. Coltivare le A431 in fiasche T75 in DMEM e 10% di FCS usando pro2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
cedure standard fino al raggiungimento del 80-90 % della confluenza
(circa 1,5x107 cellule).
Rimuovere il terreno di crescita, lavare le cellule con PBS sterile e tripsinizzare le cellule.
Raccolgliere le cellule, neutralizzare la tripsina e pellettare per centrifugazione.
Rimuovere il surnatante sopra il pellet di cellule e risospendere le cellule
in terreno.
Diluire le cellule fino a 20 ml usando terreno completo e contare le cellule.
Diluire le cellule con terreno completo fino a 200.000 cellule/ml.
Miscelare delicatamente.
Dispensare 200 μl per ogni pozzetto della piastra da 96 (40.000 cellule
per pozzetto).
Monitorare la crescita fino al raggiungimento della confluenza (tre giorni)
Riscaldare DMEM senza siero a 37°C.
Rimuovere il terreno da ogni pozzetto mediante aspirazione.
Incubare le cellule per 4-16 ore con 200 μl di DMEM preriscaldato.
Preparare in un’altra piastra da 96 pozzetti le diluizioni di EGF in 100 μl di
DMEM, come da Figura 1.
Rimuovere il terreno povero dai pozzetti con le cellule e sostituirlo con le
rispettive diluizioni di EGF in DMEM.
Incubare a 37°C per 7,5 minuti.
Preparare una nuova soluzione di fissaggio:
1 x PBS
37% formaldeide
Totale
45 ml
5 ml
50 ml
17
17. Rimuovere il terreno contenente EGF e fissare le cellule immediatamen-
18.
te con 150 μl della soluzione di fissaggio, incubare a temperatura ambiente per 20 minuti senza agitazione. La soluzione deve essere pipettata sulle pareti del pozzetto, molto lentamente in modo da impedire il
distacco di cellule.
Preparare la soluzione di lavaggio con Triton:
1 x PBS
10% Triton x100
Totale
495 ml
5 ml
500 ml
19. Rimuovere la soluzione di fissaggio.
20. Lavare 4 volte con 200 μl di soluzione di lavaggio per 5 minuti in modo
da permettere la permeabilizzazione delle cellule.
NOTE: Lavare sotto agiitazione,
non lasciare mai a secco le cellule durante i lavaggi.
21. Aggiungere ad ogni pozzetto 150 μl di LI-COR® blocking Buffer ed incubare per 1,5 ore a temperatura ambiente sotto moderata agitazione.
NOTE: Odyssey Blocking Buffer è stato ottimizzato per avere le migliori performance di
blocking. Latte in polvere o caseina sciolti in PBS possono essere usati sia per il
blocking che per le incubazioni degli anticorpi, facendo attenzione però ad usare il
latte su PVDF perché potrebbe causare un alto background. Nel caso si preferisca
usare la caseina, soluzioni al 0.1 o 0.2 % in PBS sono preferite. Non è richiesta
caseina a purezza Hammersten.
Blocking a base di latte possono interferire con la detection del segnale se si usano
anticorpi anti-goat.
Soluzioni di blocking contenenti BSA possono essere impiegate, ma in
alcuni casi possono dare livelli di background maggiori. Blocking buffer
contenenti BSA non sono generalmente consigliati, ma possono essere
usati quando l’anticorpo primario richiede l’uso di BSA come bloccante.
22. Diluire gli anticorpi in LI-COR blocking buffer come da protocollo suggerito dal fornitore e seguendo queste combinazioni:
Fosfo EGFR tyr 1045 (rabbit) e total EGFR (rabbit)
Fosfo EGFR tyr 1045 (rabbit) e total ERK2 (rabbit)
Fosfo ERK (mouse) e total ERK1 (rabbit)
Fosfo EGFR tyr 1045 (rabbit) e total ERK (mouse)
23. Aggiungere 50 μl di LI-COR Odyssey Blocking Buffer ad una serie di
pozzetti che serviranno da controllo di background dovuto all’anticorpo
secondario marcato (Figura 1).
18
PROTOCOLLI APPLICATIVI
24. Miscelare bene le soluzioni di anticorpo prima di pipettarle nei pozzetti.
25. Rimuovere il blocking buffer dai pozzetti (esclusi i controlli di back-
26.
27.
ground), per aspirazione aggiungere 50 μl della combinazione di anticorpi primari desiderata nei pozzetti rimanenti. Tutto il fondo del pozzetto deve essere ricoperto.
Incubare con l’anticorpo primario per 2 ore a temperatura ambiente,
sotto agitazione, oppure over night a 4°C senza agitazione.
Preparare una soluzione di lavaggio con Tween-20:
1 x PBS
20% Tween-20
Totale
995 ml
5 ml
1000 ml
28. Lavare la piastra con la soluzione di lavaggio contenente Tween-20
29.
30.
aggiungendo gentilmente il tampone sulla parete del pozzetto. Usare
200-500 μl per ogni lavaggio, da effettuare sotto gentile agitazione per
5 minuti.
Ripetere il lavaggio per 4 volte.
Calcolare le quantità di anticorpi secondari per completare la piastra,
e diluirli in Odyssey Blocking Buffer. Per ridurre il background aggiungere 0,2 % di Tween-20. Le diluizioni raccomandate sono tra 1:200 e
1:2000.
Goat Anti-Rabbit IRDye™680 (1:200)
Goat Anti-Mouse IRDye™800 (1:800)
31. Miscelare le due soluzioni, aggiungere 50 μl ad ogni pozzetto ed incu32.
33.
34.
bare 60 minuti a temperatura ambiente sotto agitazione. Proteggere la
piastra dalla luce durante l’incubazione.
Lavare la piastra con la soluzione di lavaggio contenente Tween-20,
usando gli accorgimenti dello step di lavaggio precedente. Lavare 4
volte.
Rimuovere la soluzione di lavaggio, tamponando la piastra su un foglio
di carta assorbente sino ad eliminare completamente qualsiasi residuo
di tampone.
Effettuare scansioni su Odyssey a 700 nm e 800 nm.
19
Risultati attesi
Misurazione e quantizzazione di Fosfo-EGFR
normalizzato verso EGFR totale.
nm
Background Resting 0.2
0.4
0.8 1.6 3.2 6.25 12.5 25
50
100
50
100
Two-color display of both
700 and 800 nm images
700 nm image
(phosphorylated ERK)
800 nm image
(total ERK)
Fosfo-EGFR normalizzato
verso EGFR totale
A 800 nm è stato acquisito EGFR
totale (come normalizzatore), a
700 nm invece è stato acquisito
Curva dose-risposta di cellule
A431 al Epithelial Growth Factor EGFR fosforilato. Le cellule usate
(EGF) misurato con un anticorpo come controllo di background
specifico per il recettore all’EGF sono state incubate con
fosforilato (EGFR, tyr1045).
anticorpi secondari, ma non
L’immagine rappresenta i risultati con anticorpi primari. Il grafico
su una piastra da 96 pozzetti
rappresenta i dati quantitativi
scansionata a 700 nm 800 nm.
normalizzati, espressi in valore %.
Misurazione e quantizzazione di Fosfo-EGFR
normalizzato verso ERK totale.
nm
Background Resting 0.2
Two-color display of both
700 and 800 nm images
700 nm image
(phosphorylated EGFR)
800 nm image
(total ERK.)
20
PROTOCOLLI APPLICATIVI
0.4
0.8 1.6 3.2 6.25 12.5 25
Fosfo-EGFR normalizzato
verso ERK totale
A 800 nm è stato acquisito EKR
totale (come normalizzatore), a
700 nm invece è stato acquisito
Curva dose-risposta di cellule
A431 al Epithelial Growth Factor EGFR fosforilato. Le cellule usate
(EGF) misurato con un anticorpo come controllo di background
specifico per il recettore all’EGF sono state incubate con
fosforilato (EGFR, tyr1045).
anticorpi secondari, ma non
L’immagine rappresenta i risultati con anticorpi primari. Il grafico
su una piastra da 96 pozzetti
rappresenta i dati quantitativi
scansionata a 700 nm 800 nm.
normalizzati, espressi in valore %.
Misurazione e quantizzazione di Fosfo-ERK
normalizzato verso ERK totale.
nm
Background Resting 0.2
0.4
0.8 1.6 3.2 6.25 12.5 25
50
100
Two-color display of both
700 and 800 nm images
700 nm image
(phosphorylated ERK)
800 nm image
(total ERK.)
Fosfo-ERK normalizzato
verso ERK totale
A 800 nm è stato acquisito ERK
totale (come normalizzatore), a
700 nm invece è stato acquisito
Curva dose-risposta di cellule
A431 al Epithelial Growth Factor EKR fosforilato. Le cellule usate
(EGF) misurato con un anticorpo come controllo di background
specifico per ERK fosforilato
sono state incubate con
(tyr204). L’immagine rappresenta anticorpi secondari, ma non
i risultati su una piastra da 96
con anticorpi primari. Il grafico
pozzetti scansionata a 700 nm
rappresenta i dati quantitativi
800 nm.
normalizzati, espressi in valore %.
21
Misurazione e quantizzazione di Fosfo-ERK
e Fosfo-EGFR.
nm
Background Resting 0.2
0.4
0.8 1.6 3.2 6.25 12.5 25
Two-color display of both
700 and 800 nm images
700 nm image
(phosphorylated ERK)
800 nm image
(total ERK.)
Fosfo-ERK e Fosfo-EGFR
Curva dose-risposta di cellule
A431 al Epithelial Growth Factor
(EGF) con misurazione specifica
di fosfo-EGFR (tyr1045) e fosfoERK (tyr204) simultanea. A 700
nm è stato acquisito il fosfo-EGFR,
mentre a 800 nm è stato acquisito
fosfo-ERK. Le cellule usate come
controllo di background sono state
incubate con anticorpi secondari,
ma non con anticorpi primari.
22
PROTOCOLLI APPLICATIVI
50
100
PUBBLICAZIONI
IMAGING INFRAROSSO
L'analisi quantitativa dell'espressione proteica
Near-Infrared Technology & Optical Agents
for Molecular Imaging
D. Michael Olive, Ph.D. – Molecular Biology Department – LI-COR Biosciences
Abstract
Optical imaging is a rapidly developing biomedical technology that enables the examination
of cellular processes in the context of a living animal. While several optical imaging modalities
employing fluorescent proteins or bioluminescent reporter systems have shown utility in life
science research, targeted ligands labeled with near infrared emitting fluorochromes have the
additional potential to translate to human clinical use. Described here is a brief overview of the
optical imaging technologies currently in use with a particular focus on the use of ligand-targeted near infrared fluorochromes as imaging agents.
Introduction
Optical Imaging consists of several
technologies which can be used to noninvasively interrogate an animal model for
the progression of a disease, determine
the effects of drug candidates on the target
pathology, assess the pharmacokinetic
behavior of a drug candidate, compare
candidate drugs for target binding affinity,
and develop biomarkers indicative of
disease and treatment outcomes. Optical
Imaging is comprised of three approaches
which offer the potential for high sensitivity
and good spatial resolution.
Bioluminescent imaging is an indirect
technique based on the expression
of firefly luciferase from recombinant
plasmids inserted into hybrid cell lines
that can be transplanted into animals. In
some cases the gene is constitutively
expressed and the introduction of luciferin,
either injected or inhaled, allows the
production of light in the target cells. In
a second case, the luciferase gene can
be activated by chemical induction of the
promoter controlling the gene’s expression
with the subsequent generation of light
upon contact with luciferin. This approach
requires the creation of modified cells, and
analysis is limited to cells expressing the
luciferase gene. Thus it is not translatable
to clinical practice.
In a second approach, cells engineered to
express fluorescent proteins can be used
to mark tumors. The recombinant tumor
cells can be implanted into animals and,
following excitation with an appropriate
light source, the fluorescence from the
expressed fluorescent proteins can be
detected by means of a CCD camera. The
excitation and emission wavelengths of
commercially available fluorescent proteins
are generally in the visible region of the
spectrum. As discussed in this publication,
this region can be compromised by
tissue autofluorescence and non-specific
background. Also, this method requires
transgenic cell lines and is thus limited
in its ability to detect a variety of potential
targets. Similar to bioluminescent imaging,
this method cannot translate to the clinic.
A more flexible and direct approach
employs
targeted
imaging
agents
consisting of antibodies, receptor binding
ligands, small molecules, or peptides
labeled with fluorochromes. The fluorescent
labels can be visualized by excitation with
an appropriate light source and the emitted
photons captured via a CCD camera or
other optical detector.
For fluorescent imaging, there are
generally three parameters which are used
to characterize the interaction of photons
with tissues. The three processes are light
23
absorption, light scattering, and fluorescent
emission. A fundamental consideration in
optical imaging is maximizing the depth
of tissue penetration. Absorption and
scattering of light is largely a function of
the wavelength of the excitation source1.
In general, light absorption and scattering
decreases with increasing wavelength2.
Below 700 nm, tissue absorption results
in small penetration depths of only a few
millimeters1. Thus in the visible region of
the spectrum, only superficial assessment
of tissue features is possible.
The light absorption is due to oxyand deoxyhemoglobin, melanin, lipid,
and other compounds found in living
tissue2,3,4. These compounds cause tissue
autofluorescence throughout the visible
spectral range up to approximately 700
nm5,6. Because the absorption coefficient
of tissue is considerably smaller in the near
infrared region (700 nm-900 nm), light can
penetrate more deeply into the tissues to
depths of several centimeters3,7,8.
A key to enabling optical imaging has
been the development of suitable NIR
fluorochromes with high molar extinction
coefficients, good quantum yields and
low non-specific tissue binding. There are
several commercially available candidate
fluorochromes which can be used for
optical imaging, including IRDye® 800CW,
IRDye 680, IRDye 700DX, Cy®5.5, and
Alexa Fluor® 750. Quantum dots have been
used; however, their size often precludes
efficient clearance from the circulatory and
renal systems and there are questions
about their long-term toxicity4.
Figure 1.
Molar extinction coefficient
characteristics of water,
hemoglobin and oxygenated
hemoglobin.
Emission wavelengths are shown
for Cy5.5 and IRDye 800CW.
24
PUBBLICAZIONI
A number of studies have been published
using NIR dyes. Two of the dyes commonly
used for optical imaging are Cy5.5 and
IRDye 800CW. Cy5.5 has been used in
the past primarily due to the lack of other
candidate dyes suitable for imaging.
Cy5.5 has excitation/emission maxima at
675 nm/694 nm, making it a borderline
candidate labeling agent1,3. In contrast, a
recently developed fluorochrome, IRDye
800CW, has its excitation/emission maxima
at 785 nm/810 nm precisely centered in
the region known to give optimal signal to
background in optical imaging (see Figure
1)1,8.
In a cell based assay system using IRDye
800CW labeled secondary antibodies
to assess cell signaling pathways, IRDye
800CW was shown to yield superior
signal-to-background ratios and enabled
quantification of low levels of protein
phosphorylation9.
In contrast, the signal to background using
Cy5.5-labeled secondary antibodies was
too low to allow its use in the assay (data
not shown). Further experiments showed
that Cy5.5 exhibited a high level of nonspecific binding to cells (data not shown).
This observation, coupled to the fact that
Cy5.5 is outside the optimal NIR region,
makes it less suitable for imaging studies
requiring high signal to background.
The performance of IRDye 800CW has been
compared to radiochemical detection in
animal studies. Using gamma scintigraphy
and NIR imaging, Houston et al. compared
the ability of a cyclopentapeptide duallabeled with111 indium and IRDye 800CW to
image ανβ3-integrin positive melanoma
xenografts10. The tumor regions were
clearly delineated by optical imaging of
the IRDye 800CW signal. In contrast, the
tumor boundaries could not be identified
by scintigraphy due to high noise levels.
Figure 2.
Tumor imaging with IRDye 800CW-EGF. A SCID
mouse bearing an orthotopic prostate tumor was
injected with 1 nmole IRDye 800CW-EGF and
imaged on an Odyssey Infrared Imaging System.
We have successfully used IRDye 800CW
conjugated to epidermal growth factor
(IRDye 800CW-EGF) as an optical agent
for imaging tumor progression11.
Figure 2 shows an example of a prostate
tumor-bearing SCID mouse injected with
IRDye 800CW-labeled EGF.
The IRDye 800CW-EGF showed good
sensitivity with very low background from
autofluorescence or non-specific binding.
For animal imaging, targeted NIRlabeled ligands can be used to visualize
virtually any pathology without the need
for engineered cell lines as required for
imaging by bioluminescence or fluorescent
proteins. In addition, NIR optical imaging
has a further advantage in that it has the
potential to translate into the clinic. Several
NIR imaging instruments for use on
humans are currently under development.
De Grand and Frangioni have described a
prototype NIR optical imaging system for
use with NIR fluorochrome-labeled optical
agents in non-invasive intraoperative
imaging procedures12. The authors
envision the system being eventually
used for image-guided cancer resection
with real-time assessment of surgical
margins, sentinel lymph node mapping,
25
and intraoperative mapping of normal and
tumor vasculature. Furthermore, Gurfinkel
et al., Hawrysz and Sevick-Muraca, and
Chen et al., have described NIR-based
imaging instruments directed at the early
detection of breast cancer 3,13,14. These
instruments potentially could be used for
guiding fine needle biopsies and sentinel
lymph node monitoring during surgery.
References
There are several biological barriers that
should be taken into consideration when
using NIR dye-labeled optical probes.
The probe must be able to reach its
target in sufficient concentration and with
sufficient binding affinity that it can be
imaged. In this respect, optical probes are
similar to a pharmaceutical agent in that
considerations of absorption, distribution,
metabolism, excretion, and toxicity need to
be evaluated.
4. Frangioni, J. V. 2003. Curr. Opinion. Chem.
Biol. 7:626-634.
In addition to non-specific binding,
trapping, rapid excretion, and metabolic
effects, there are delivery barriers to be
overcome. For example, the size and
characteristics of the dye labeled ligand
may prevent it from crossing the bloodbrain barrier. However, the combination
of an NIR labeling agent such as
IRDye 800CW and NIR-based imaging
instruments used for both small animal and
clinical imaging has the potential to provide
both good spatial resolution and sensitive
detection of targeted molecules. In the
future, NIR imaging technology should
augment current imaging technologies and
provide a means of characterizing disease
processes and monitoring therapeutic
efficacy, enabling earlier detection through
the identification of molecular biomarkers
in both the research laboratory and the
clinic.
26
PUBBLICAZIONI
1. Licha, K. 2002. Topics Curr. Chem. 222:129.
2. Tromberg, B. J., Shah, N., Lanning,
R., Cerussi, A., Espinoza, J., Pham, J.,
Svaasand, L., and Butler, J. 2000. Neoplasia
2:26-40.
3. Hawryz, D. J. and Sevick-Muraca E. M. 2000.
Neoplasia 2:388-417.
5. Andersson-Engels, S. and Wilson, B. C.
1992. J. Cell Pharmacol. 3:48-60.
6. Wagnières, G. A., Star, W .M., and Wilson,
B. C. 1998. Photochem. Photobiol. 68:603632.
7. Grosenick, D., Wabnitz, H., Rinneberg, H.,
Moestra, K. T., and Schlag, P. M. 1999. Appl.
Optics. 38:2927-2038.
8. Shah, K. and Weissleder. 2005. J. Amer.
Soc. Exp. Neurother. 2:215-225.
9. Chen, H., Kovar, J., Sissons, S., Cox, K.,
Matter, W., Chadwell, F., Luan, P., Vlahos, C.,
Schutz-Geschwender, A., and Olive, D. M.
2005. Anal. Biochem. 338:136-142.
10. Houston, J. P., Ke, S. Wang, W., Li, C., and
Sevick-Muraca, E. M. 2005. J. Biomed.
Optics. 10:054010-1-11.
11. Kovar, J. L., Johnson, M. A., Volcheck, W. M.,
Chen, Jiyan, and Simpson, M. A. 2006. The
American Journal of Pathology. 169:14151426.
12. De Grand, A. M. and Frangioni, J. V. 2003.
Technol. Cancer Res Treatment. 2:1-10.
13. Gurfinkel, M., Ke, S., Wen, X., Li, C., SevickMuraca, E. M. 2003. Disease Markers.
19:107-121.
14. Chen, Y., Intes, X., and Chance, B. 2005.
Biomed. Instru. Technol. 39:75-85.
A systematic approach to the development of fluorescent contrast
agents for optical imaging of mouse cancer models
Joy L. Kovar1, Melanie A. Simpson2, Amy Schutz-Geschwender1, and D. Michael Olive1*
1LI-COR Biosciences
2Department of Biochemistry, University of Nebraska
Available online at www.sciencedirect.com
Originally published in Analytical Biochemistry, Vol. 367, #1, August 1, 2007, Pages 1-12.
Abstract
Optical imaging is a rapidly developing field of research aimed at non-invasively interrogating animals for
disease progression, determining the effects of a drug on a particular pathology, assessing the pharmacokinetic behavior of a drug, or identifying molecular biomarkers of disease. One of the key components of
molecular imaging is the development of specific, targeted imaging contrast agents to assess these biological processes. The development of robust fluorochrome-labeled optical agents is a process that is
often underestimated in terms of its complexity. Although many studies describe the use of these agents,
guidelines for their development and testing are not readily available. This review outlines some of the
general principles that are important when developing and using fluorochrome-labeled optical contrast
agents for oncology investigations in animals.
*Corresponding author:
D. Michael Olive
Vice President, Science and Technology
LI-COR Biosciences
4647 Superior Street
Lincoln, NE 68504
Tel: 402-467-0762
Fax: 402-467-0819
Email: [email protected]
27
Page 2 – A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models
In the last decade, our increased understanding of the molecular basis of cancer has led to the development of novel
targeted strategies for specific inhibition of cancer signaling pathways that control growth, proliferation, apoptosis,
and angiogenesis. Several monoclonal antibody-based
therapeutics and small molecule drugs have received clearance for use as human therapeutics [1]. However, among
these successes are many candidate drugs that have failed
in clinical trials despite promising pre-clinical results [2].
The development of targeted therapeutics is expensive and
time consuming. In their Critical Path Initiative, the United
States Food and Drug Administration emphasized the need
for more effective tools to facilitate the rapid development
of improved cancer therapeutics. One such tool is the use
of targeted molecular optical imaging probes or contrast
agents to visualize the underlying processes in cancer.
Optical imaging, also known as molecular imaging, is a rapidly developing field of research aimed at non-invasively
interrogating animals for disease progression, evaluating
the effects of a drug, assessing the pharmacokinetic behavior of a drug, or identifying molecular biomarkers of
disease. A prerequisite of molecular imaging is the development of specific, targeted imaging contrast agents to assess these biological processes. Several optical aids have
shown great utility in animal studies, including bioluminescence, fluorescent proteins, and fluorochrome-labeled
agents. However, only the latter have the advantage of
being potentially relevant to human clinical applications.
The complexity of developing robust fluorochrome-labeled
optical agents is often underestimated. Many studies describe the use of these agents, but guidelines for their development and testing are not readily available. The
purpose of this review is to outline some of the considerations for developing and using fluorochrome-labeled optical contrast agents in animals. For simplicity, we have
focused on the use of organic fluorochromes as labeling
agents. These types of probes are generally the most
straightforward to develop and have the greatest potential
for translation to human clinical use. Nanoparticles such
as quantum dots, while useful for some animal studies, are
hampered by clearance issues and toxicity and will not be
specifically discussed. However, the principles described
here are generally applicable to any fluorescent optical imaging agent.
Principles of fluorescence imaging
The use of fluorochrome-labeled optical agents such as labeled antibodies, receptor-binding ligands, small molecules, peptides, and activatable probes offers a flexible and
direct imaging methodology. The fluorescent labels can be
visualized by excitation with an appropriate light source
and capture of the emitted photons with a CCD camera or
other optical detector. Several commercially available imaging systems enable visualization of these probes in mice.
These include systems from LI-COR Biosciences
(www.licor.com),
CRI
(www.cri-inc.com),
Kodak
(www.kodak.com) and Xenogen (www.xenogen.com).
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PUBBLICAZIONI
In fluorescent imaging, there are generally three parameters used to characterize the interaction of photons with tissues: light absorption, light scattering, and fluorescent
emission. One of the most important considerations in optical imaging is maximizing the depth of tissue penetration.
Absorption and scattering of light are largely a function of
the wavelength of the excitation source [3]. Light is absorbed by endogenous chromophores found in living tissue, including hemoglobin, melanin, and lipid [3-7]. In
general, light absorption and scattering decrease with increasing wavelength. Below ~700 nm, these effects result
in shallow penetration depths of only a few millimeters [3].
Thus, in the visible region of the spectrum, only superficial
assessment of tissue features is possible. Above 900 nm,
water absorption can interfere with signal-to-background
ratio. Because the absorption coefficient of tissue is considerably lower in the near infrared (NIR) region (700-900
nm), light can penetrate more deeply, to depths of several
centimeters [3-6]. Fluorochromes with emissions in the NIR
are not hindered by interfering autofluorescence, so they
tend to yield the highest signal-to-background. The combination of increased depth of penetration and reduced autofluorescence makes NIR fluorochromes ideally suited for
optical imaging in small animals, and potentially in humans
as well (Figure 1).
Figure 1. Schematic representation of the region of optimal signal-to-background ratio in tissue. Hemoglobin
can interfere below 700 nm, while water interferes
above 900 nm. The excitation and emission regions for
several dyes commonly used in optical imaging are also
indicated.
Near infrared fluorochromes
A key to enabling optical imaging has been the development of suitable NIR fluorochromes. Important criteria for
effective optical imaging fluorochromes include: excitation
and emission maxima in the NIR between 700-900 nm; high
quantum yield [3, 5, 6, 7]; chemical and optical stability; and
suitable pharmacological properties including aqueous solubility, low non-specific binding, rapid clearance of the free
dye, and low toxicity. The dyes most commonly used for
fluorescent optical imaging are listed in Table 1.
The cyanine dye Cy5.5 has been used frequently for in vivo
imaging. The excitation/emission maxima (675 nm/695 nm)
A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models – Page 3
Table 1
Excitation and emission maxima of fluorescent
dyes commonly used for optical imaging
Fluorophore
Cy5.5
Alexa Fluor 680
IRDye 680
Alexa Fluor 700
IRDye 700DX
Alexa Fluor 750
Cy7
IRDye 800CW
Excitation
Max (nm)
Emission
Max (nm)
675
679
680
702
689
749
749
774
695
702
709
723
700
775
775
805
are close to the NIR region, yielding acceptable signal-tobackground. Other dyes with excitation and emission
maxima in this region include IRDye® 700DX, IRDye 680,
Alexa Fluor® 700 and Alexa Fluor 680. Although excitation
and emission wavelengths of these dyes are maximal in
the more favorable red region, they do not give the optimal performance that can be achieved by moving farther
into the NIR. Thus they are best used in situations when
the highest sensitivity is not required. However, the pthalocyanine dye IRDye 700DX has properties that may make it
attractive for imaging. IRDye 700DX is considerably less
sensitive to photobleaching than many organic fluorochromes; Peng et al. [8] have shown that IRDye 700DX is
100 times more photostable than Alexa Fluor 680 and
Cy5.5. In addition, members of the pthalocyanine dye class
have been used for photodynamic therapy in the treatment
of several types of cancer [9]. The photodynamic characteristics relevant to tumor therapy require prolonged exposure to the light source and therefore would not interfere
with tumor biology during the short exposures used for imaging. Therefore, pthalocyanines have the potential to
serve not only as imaging agents but also therapeutics.
The most widely used dyes with true NIR character include
IRDye 800CW, Cy7, and Alexa Fluor 750. The excitation/emission ranges for these dyes are shown schematically in Figure 1. In particular, IRDye 800CW has
excitation/emission maxima of 774 nm/789 nm, which are
centered at the optimal wavelength for in vivo imaging.
This dye has been shown to be superior in performance to
Cy5.5 in terms of signal-to-background [3, 7, 10].
Targets and ligands
Many targeted optical probes have been described in the
literature. Targets include cell surface receptors, metabolic
pathways, hormone receptors, apoptotic markers, and enzymatic activities [11]. High affinity probes may be developed by rational investigation, combinatorial chemical
synthesis, or phage display. An effective agent reaches the
target at a sufficient concentration and/or is retained there
for a sufficient length of time to be visible at the time of imaging. Obstacles such as rapid excretion, metabolism, non-
specific binding, and physical barriers to the agent reaching
the target must be overcome in order for a targeted optical
agent to function robustly.
Delivery barriers present the greatest obstacle but can be
circumvented. One approach is to take advantage of normal cellular transport and endocytic processes by targeting surface receptors or transport pathways that internalize
the optical agent. Growth factor receptors are an example
in which the binding of a fluorochrome-labeled agent stimulates internalization via endocytosis. This has the added
benefit of amplifying the fluorescent signal, since the fluorochrome will accumulate in the target cells. A second approach is to incorporate a peptide membrane translocation
signal into the optical agent such that active transport of
the imaging agent across the cell membrane results. Signal peptides such as the HIV tat peptide have been successfully used to transport nanoparticles into cells [12].
Non-specific binding is another critical issue for noninvasive tumor imaging. In vivo, the inability to eliminate unbound ligand can cause low signal-to-background. In
addition, non-specific binding or retention of the optical
probe will yield false positive results. Careful assessment
of the optical agent clearance pattern and verification of
specific signal by competition experiments can address this
issue.
Antibody conjugates
Many of the first fluorochrome-conjugated targeted imaging agents were antibodies. For example, indocyanine-conjugated monoclonal antibodies against cells derived from
a squamous cell carcinoma have been used to image A431
cell xenografts in nude mice [13]. Detection of the
xenografts was sensitive and specific. Cy3, Cy 5, and Cy5.5conjugated monoclonal antibodies have been used to direct SSEA-1 for detection of MH-15 teratocarcinoma
xenografts [14]. In this study, fluorescence did concentrate
in the tumor, but significant background from the conjugate was observed in the kidneys and bladder. The NIR dye,
Cy5.5, appeared to yield the best signal-to-background.
Lastly, minibodies directed against the extra-domain B of fibronectin and conjugated to Cy7 have been used to successfully image atherosclerotic plaques in a mouse model
[15].
Although antibody conjugates have been successfully
used, they have several undesirable features, primarily due
to the size of the antibody. Larger molecules, such as antibodies, can elicit an adverse immune response from the
host animal, and their long half-life in the blood results in
high background fluorescence and long clearance times
[16, 17]. In addition, large biomolecules are often taken up
preferentially by the liver, precluding imaging of liver-proximal organs [18]. Finally, in order for the contrast agent to
effectively penetrate to the target site, it must diffuse from
the vasculature to the site of the pathology; larger molecules show very poor diffusion characteristics which may
prevent them from reaching the target site [19].
Tumor surface proteins
Tumor surface proteins offer diverse possibilities for targeting of optical probes. An excellent example is a recep-
29
Page 4 – A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models
tor-binding ligand. Growth factors are a popular choice for
optical imaging agents because in addition to high affinity
targeting, the ligand and its fluorescent label are often internalized by the normal endocytic pathway, amplifying the
signal in the tumor cells. Fluorochrome-labeled epidermal
growth factor (EGF) has been a versatile tumor imaging
agent because the epidermal growth factor receptor (EGFR)
is overexpressed on the surface of many types of tumor
cells [20, 21, 22, 23, 24]. In a study measuring the diffusion
of small molecules across the extracellular space in rat
brains, Thorne et al. [25] showed that EGF labeled with Oregon Green 514 could be used as a reporter. Ke et al. [26]
used Cy5.5-labeled EGF to target human breast tumor cells
implanted in mice. The EGF-Cy5.5 accumulated specifically
in the tumors and uptake was blocked by pretreatment of
animals with C225 anti-EGFR monoclonal antibody (cetuximab).
Similarly, we have demonstrated the utility of EGF labeled
with IRDye 800CW for analysis of orthotopic prostate tumors in mice [27, 28]. IRDye 800CW EGF accumulated
specifically in the tumors, and metastatic spread of the primary orthotopic tumor to the para-aortic lymph nodes was
detected. EGF is known to stimulate tumor growth, and
this is an important concern for its use as an imaging agent.
Comparison of tumors excised from animals injected with
labeled EGF only at the study endpoint to tumors excised
from mice that had been injected at weekly intervals over
a six week period demonstrated no stimulation of tumor
growth by the fluorochrome-conjugated ligand used in this
longitudinal study [27].
Endostatin, a 20 kD fragment of collagen XVIII, is a potent
inhibitor of angiogenesis. Using Cy5.5-labeled endostatin,
Citrin et al. [29] were able to demonstrate that the labeled
optical agent bound specifically to tumor xenografts in
C57BL mice suggesting that the anti-angiogenic effect of
endostatin is due to its action directly on the tumor cells
rather than a general anti-angiogenic effect. The Cy5.5-endostatin bound specifically to the tumor and the signal persisted for up to seven days post-intraperitoneal injection
[29].
Apoptosis plays an important role in a number of disease
pathologies, particularly cancer. One of the earliest markers of apoptosis is the externalization of phosphatidylserine. Annexin V, a 36 kD protein, exhibits high affinity for
phosphatidylserine and has been used to detect apoptosis
in vivo. Petrovsky et al. [30] and Ntziachristos et al. [31]
demonstrated the utility of Cy5.5-labeled annexin V for detection of apoptosis in a mouse tumor model. This marker
may be useful in studying the anti-proliferative effects of
chemotherapeutic agents on a variety of cancers.
Somatostatin receptors and their ligands have been used
as a targeting system for tumor imaging and radiotherapy
of cancer for over 15 years. Radiolabeled synthetic analogues of somatostatin have been used to successfully
image gastric or pancreatic tumors as well as small cell
lung cancer (SCLC) [32, 33]. SCLC is a major cause of death
in western countries. The substitution of fluorochrometagged somatostatin and several analogues has enabled
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PUBBLICAZIONI
imaging of human H69 SCLC tumor xenografts in mice
using fiber-optic spectrofluorimetry. Thus, near-infrared
conjugated peptides may have significant clinical impact
on tumor detection by endoscopy, mammography, and intraoperative imaging.
Peptides and Small Molecules
Because of their small size, convenience of handling and
attractive pharmacokinetic properties, peptides are useful
agents for in vivo imaging. Peptides targeting integrins, a
family of cell surface receptors that broadly regulate tumor
growth, metastasis, and angiogenesis, have been successful agents for imaging neovascular density [34-41]. Chen
et al. [37] and Achilefu et al. [41] used an NIR peptide conjugate, arginine-glycine-aspartic acid (RGD), targeting αvβ3
integrin, to detect and image tumor xenografts and to monitor angiogenesis. Tumor uptake of the Cy-5.5 RGD peptide
was specific and could be blocked by pre-injection with unlabeled RGD. Houston et al. [42] used an RGD peptide doubly labeled with 111indium and IRDye 800CW to directly
compare NIR optical imaging with scintigraphy. The authors found the signal-to-background ratio significantly
higher for IRDye 800CW than for the radioactive label.
In general, small molecule and peptide imaging agents
clear the system quickly, translating to a reduction in fluorescent background when imaging. In all cases described
above, the agents cleared through the kidneys without
pooling in the liver. The small size also greatly reduces the
possibility of an adverse immune response. Finally, fluorochrome-labeled small molecules and peptides penetrate
to the target efficiently because of their increased ability to
diffuse from the vasculature.
Activatable probes
The last category of fluorescent agents, activatable probes
or “molecular beacons” [43, 44], specifically yield a fluorescent signal only when activated by an enzyme target.
Most activatable probes are protease substrates. Protease
levels are elevated in the extracellular space of many tumors, where they play a role in invasion and metastasis
[45-49], and present a physically accessible target. Typically, these probes contain multiple NIR fluorochromes coupled to peptide sequences that can be cleaved by the
protease. The proximity of the fluorochromes to each other
results in quenching that is relieved upon cleavage by the
target protease to generate a fluorescent signal. Use of fluorochrome/quencher pairs separated by the peptide target
sequence has also been reported.
Proteases that have been targeted by activatable probes include cathepsins, matrix metalloproteinases (MMPs),
prostate-specific antigen, thrombin, caspase-3, and interleukin-1β converting enzyme [50]. An example of the signal sensitivity that can be achieved using these agents is
the visualization of MMP-2 activity [50, 51, 52]. In vitro, the
authors observed an 850% increase in NIR fluorescence intensity when the probe was cleaved and specific activation
could be blocked by MMP-2 inhibitors. MMP-2 positive
human fibrosarcomas were visualized and differentiated
from MMP-2 negative mammary adenocarcinomas using
this probe.
A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models – Page 5
Developing an Optical Imaging Agent
Below, we will discuss some of the critical parameters involved in developing, validating, and using an optical imaging agent. We present an overview of the process as it
applies to tumor imaging, using as an example the
IRDye 800CW EGF imaging agent we recently developed
[27, 28]. Again, although we use this agent as an example,
the principles described will be applicable to any dye-conjugate optical agent.
Conjugating probes with NIR dyes
Development of an optical targeting agent begins with covalent attachment of an NIR dye to the targeting compound. Many dyes are available with an N-hydroxysuccinimidyl (NHS) ester, which is activated for simple onestep coupling to free amines. NHS esterified NIR dyes may
be purchased in bulk or in pre-packaged labeling kits.
Methods for removal of unreacted dye may include HPLC,
FPLC, or dialysis. Purification will be dictated by the molecular weight and chemical properties of the conjugates.
For in vivo imaging applications, the dye/protein ratio of
the conjugate may affect biological or biochemical activity
of the protein, signal-to-background ratio, clearance, and
biodistribution [52]. The optimal ratio of coupling is unique
to each targeting agent. For example, Ntziachristos et al.
[31] found that annexin V labeled with Cy5.5 at a dye/protein ratio of 1 retained its ability to bind phosphatidylserine
on the surface of cells induced for apoptosis, while the
same molecule labeled at a dye/protein ratio of 2.4 had lost
its binding capability.
If the optical imaging agent is based on a commercially
available reagent such as an antibody or receptor ligand,
differences in preparation and purification may impact performance. We conjugated IRDye 800CW to human recombinant EGF from five different commercial sources, at
equivalent dye/protein ratios, and evaluated relative signal
intensity by In-Cell Western [28]. As shown in Fig. 2, there
were considerable differences in the amount of EGF bound
to the cells. Variations in signal strength measured in this
fashion have the potential to predict probe performance
in vivo as we have demonstrated [28].
Testing the performance and specificity of an
optical imaging agent in vitro
is an important prelude to animal studies. Specificity can
be demonstrated on cells in culture or in suspension by
blocking the target with an antibody or by competition with
the unlabeled agent. In targeting somatostatin receptors,
Becker et al. [53] used a flow cytometric assay to quantify
binding of the agent. As mentioned previously, Ntziachristos et al. [34] used whole cell competition assays to demonstrate both probe specificity and binding affinity. Another
group used a radioactive displacement assay [54].
125I-labeled echistatin, an integrin-binding ligand, was
added to integrin-expressing cell cultures and allowed to
bind. The degree of displacement of the radioactive echistatin by Cy7-labeled RGD peptide was used as a measure of
specificity and binding affinity.
We have used the In-Cell Western (ICW) or cytoblot to evaluate IRDye 800CW EGF for binding and specificity prior to
actual testing in mice (27, 55). In this assay, PC3M-LN4 and
22Rv1 human prostate adenocarcinoma cells were cultured
in microtiter plates and treated with serial dilutions of
labeled EGF (Fig. 3A) to verify high affinity binding of EGFRtargeted dye relative to the low binding of free dye alone
(Fig. 3B). Specificity was then established in two ways: by
blocking access of EGF to its receptor with the anti-EGFR
monoclonal antibody C225 (also known as cetuximab or
Erbitux, Fig. 3C); and by competition with unlabeled EGF
(Fig. 3D). Fluorescence of the microplate was quantified by
NIR imaging, and a DNA stain was used to normalize variations in cell number.
Characterization of the targeting agent in a cell-based assay
can simplify probe development. While success in a cellbased assay format does not guarantee performance
in vivo, failure in this assay is generally predictive of failure
in the animal. In addition, the competition assays developed may subsequently be useful for demonstrating specificity in animal experiments.
Animal Care and Use
All research animals must be handled according to protocols that comply with the animal care and use regulations
of the country and institution where the research will be
performed. In the United States, these regulations are described in a document compiled by the National Institutes
of Health Organization for Lab Animal Welfare, available at
http://grants.nih.gov/grants/olaw/GuideBook.pdf.
Validation of targeting agent efficacy and specificity in vitro
Figure 2. Binding activity of recombinant EGF from
different commercial sources. EGF from five sources
was conjugated to IRDye 800CW at similar dye/protein ratios and each was evaluated for its ability to
bind confluent A431 monolayers by In-Cell Western
as we have previously described [27].
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Page 6 – A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models
Figure 3. Binding specificity of IRDye 800CW EGF for cultured PC3M-LN4 and 22Rv1 cells. Monolayer cells in
96-well microplates were incubated with increasing concentrations of IRDye 800CW EGF (A) or unconjugated,
non-reactive IRDye 800CW (B) and normalized with TO-PRO-3 staining. To demonstrate EGF receptor-targeting
specificity, cells were incubated with 70 nM IRDye 800CW EGF in the presence of increasing concentrations of
either C225 blocking antibody (C), or unlabeled EGF (D). The 800 nm signal, normalized to the 700 nm control,
is plotted as the mean ± SD of three replicate wells (27). This figure is reproduced with permission from the
American Journal of Pathology.
Considerations for tumor model selection
An ideal tumor model system exhibits minimal background
interference. Although much of the autofluorescence in the
animal is ameliorated by imaging at NIR wavelengths,
some anatomic regions inherently maintain higher fluorescent signals. For example, natural fluorescence from
compounds in the animal’s diet accumulates and can be
visualized in the abdominal cavity. Organs involved in
clearance of the dye, such as the liver and kidney, may also
accumulate signal. Tumors arising in areas remote from
these organs are detected with less ambiguity and higher
sensitivity. In the case of subcutaneous xenografts, the
placement of transplanted tissue in the flank, shoulder, or
leg of the animal can minimize these interfering factors.
The model system selected will depend on the aims of the
study. Transgenic, chemically induced, and spontaneously
arising mouse models are available that recapitulate many
aspects of the genesis, progression, and clinical course of
human cancers. The National Cancer Institute of the United
States has organized a Cancer Models Database (caMOD)
to facilitate identification of appropriate models for cancer
experimental design (https://cancermodels.nci.nih.gov/
camod/login.do;jsessionid=D8B27DC9B409DA8CC37CE041
50EBAABB).
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PUBBLICAZIONI
Three strains of immunodeficient mice are commonly used
in tumorigenesis and metastasis research with human cell
lines: nude, SCID and Rag1. The nude mouse (athymic;
nu/nu) has a disruption in the Foxn1 gene, resulting in an
absent or deteriorated thymus gland, diminished T cell
numbers, and a severely impaired cellular immune system
(56). The resultant hairless phenotype also makes the nude
mouse ideal for optical imaging, since animal hair blocks
and scatters light. Over time, these animals may regain
partial cellular immune function so nude mice may not be
the best host for longer-term studies of tumor biology.
SCID (severe combined immunodeficiency) mice lack both
mature T and B lymphocytes [57], and are an effective alternative to nude mice in cases where the partial immune
system of the nude mouse presents a problem. The Rag1
mouse also lacks T and B lymphocytes, and is not able to
undergo V(D)J recombination. Thus, it fails to produce
T-cell receptors and immunoglobulin molecules for antigen
identification [58, 59, 60]. All three mouse models bear
phenotypic and background strain characteristics that may
impact a research project.
Immunocompromised mice require Specific Pathogen Free
(SPF) handling to avoid introducing infections. Institutional
training is obligatory and includes instruction in the use of
infection barriers, sterilized food, water and bedding, dis-
A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models – Page 7
infected imaging surfaces, gloved handling and aseptic
technique.
Reducing optical interference for tumor imaging
Chlorophyll, which is often present in animal chow, absorbs
at 655 nm and 411 nm, and fluoresces at 673 nm, producing strong signal in the abdominal cavity. For optimal fluorescent imaging performance, purified food formulations
that do not contain plant products may be used. Fasting of
the animal prior to imaging has been used in some studies,
and requires prior approval from the institutional committee governing animal care.
The hairless phenotype of the nude mouse makes it an
ideal choice for NIR optical imaging, but this model may
not always be appropriate for the research objectives.
Thus, hair removal in the imaging region may be important for optimal signal detection. Animal hair interferes
with imaging by blocking, absorbing, and scattering light.
We evaluated this by implanting a tube containing
IRDye 800CW in the chest cavity of a deceased SCID
mouse, and imaging the animal before and after shaving.
Following shaving, fluorescent signal increased by >50%
(data not shown). An additional 12% signal was detected
when the animal was treated with a depilatory cream (Nair,
Church and Dwight Co., Inc.) presumably due to the removal of hair stubble.
Establishing tumors in the animal
For assays of tumorigenic and metastatic potential using
cultured human cells, cells or tissue may be implanted in
animals subcutaneously, intravenously, intraperitonally, or
orthotopically (i.e.; prostate cells implanted in mouse
prostate). These assays are called xenografts, since they
involve transplantation of cells, tissue, or organs from one
species to another.
Depending on the aggressiveness of the cell line, we have
established subcutaneous xenografts in mice by injection
of 0.5-1 106 cells in ≈100 μL cell suspension. Tumors become palpable within 7-10 days. Orthotopic injections require fewer cells (we have used 1105 cells) and reduced
injection volume (10 μL), as the anatomical structure of the
prostate is small. Tumors form in 2-3 weeks and metastasize within ≈6 weeks [27, 28].
Although background increases with the amount of probe
administered, the signal is also greater at the highest dose.
The quantified signal within the tumor, normalized to the
mean background in several irrelevant surrounding regions, allowed us to determine that the optimal specific signal occurred at the two highest doses.
Figure 4. Dose response for an IRDye 800CW conjugated optical imaging agent targeting tumors in nude
mice. Animals were injected with 1x PBS as a negative
control (A), or with 2.5 nmol (B), 5.0 nmol (C), and 10
nmol (D) of a tumor targeted IRDye 800CW conjugate.
Images were acquired 24 hr post-injection.
The route of targeting agent administration can affect its
specific uptake and non-specific clearance. Intravenous injection via the tail vein or supraorbital cavity leads to rapid
systemic dispersion (Fig.5). This method is appropriate for
targeted contrast agents that bind a ubiquitous surface receptor present at a greater concentration on tumor cells.
Uptake by the tumor cells is within the time window of
agent clearance and potential background from prolonged
exposure to the probe is minimized. Performance of an
agent that functions by incorporation into bone, however,
may be enhanced by intraperitoneal injections, which prolong exposure through slower dispersion (Fig. 5).
Probe Dosage and Administration
Tumor type may dictate the optical imaging agent selected
and its optimal parameters for use. For example, A431 epidermoid carcinoma cells express EGFR at a much higher
level than normal cells. However, HeLa ovarian carcinoma
cells have low expression of EGFR. If IRDye 800CW EGF is
used as an optical probe for both of these cell types, binding of the labeled ligand would be expected to vary dramatically.
Figure 5. Impact of administration site on optical imaging agent dispersion in mice. Images were acquired approximately 15 minutes following intraperitoneal (A) or
intravenous (B) injection of equivalent amounts of an
optical imaging agent into a nude mouse.
Evaluation of Dye and Optical Agent Clearance
An optimal dosage of the imaging agent will afford the best
signal-to-background, clearance, and imaging results. Excessively high doses will clear poorly, while a low dose
may not saturate tumor uptake. Figure 4 illustrates a dose
response to increasing concentrations of an optical agent.
Performing initial imaging time courses following injection
of the chosen targeting agent will establish the optimal
time point for sensitive tumor analysis. The time course
analysis begins with the unconjugated fluorochrome chosen for optical imaging, which should not be appreciably
33
Page 8 – A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models
Figure 6. Clearance kinetics of unconjugated IRDye 800CW. A SCID mouse was injected with 1 nmol of IRDye
800CW intravenously and monitored over time for complete clearance of the dye. Imaging times post injection are indicated on each individual image. Pseudocolored fluorescence is superimposed on the white light
image of the mouse to illustrate rapid dispersion of the dye followed by complete clearance.
Figure 7. Time course for clearance of IRDye 800CW EGF from a non-tumor-bearing mouse. A SCID
mouse was injected with 1 nmol of the EGF-conjugated optical agent intravenously and imaged at
the indicated time points over a 24 hr period. Analysis of the abdominal region showed that >90% of
the fluorescence disappeared in 24 hr, and by 48 hr (not shown) the probe had cleared completely.
retained in the animal. An example of measurement of the
clearance of IRDye 800CW is shown in Figure 6. Other dyes
may have different clearance characteristics. Secondly, a
time course of agent clearance from non-tumor-bearing
control animals using the intended dose for tumor imaging will yield a blueprint for whole-body nonspecific background to assist interpretation of tumor images. Figure 7
shows the results of a clearance experiment with
IRDye 800CW EGF in a tumor-negative mouse.
Finally, once it has been determined that the probe does
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PUBBLICAZIONI
not accumulate non-specifically, the time course is repeated with tumor-bearing animals to determine the imaging time at which the signal-to-background is greatest in
the tumor. For IRDye 800CW EGF, mice bearing PC3M-LN4
subcutaneous xenografts were imaged over a three-day
period. By 24 hr, most of the initial fluorescence was gone
from the non-tumor areas (Fig. 8). However, signal-tobackground continued to increase beyond this time point.
Optimizing the time of imaging maximizes sensitivity for
challenging applications such as detecting metastatic
spread of a tumor.
A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models – Page 9
Figure 8. Time course for accumulation of IRDye 800CW EGF in a subcutaneous tumor. Images of a
nude mouse were collected prior to injection (A), or at 20 min (B), 24 h (C), 48 h (D), and 72 h (E) following intravenous injection of the animal with 1 nmol of IRDye 800CW EGF.
Confirming Probe Specificity In Vivo
Clearance studies and optimization of timing will minimize
non-specific fluorescence, but imaging artifacts may be
misinterpreted nonetheless. For example, an apparent
tumor detected in the liver upon targeting with an antibody
probe that pooled in the liver should be confirmed by a
competition test. One method is to pre-inject tumor-bearing animals with an excess (for example, 100-fold) of the
unlabeled form of the optical agent. The labeled agent is injected shortly thereafter. Probe specificity is reflected as a
decrease in the total fluorescence signal in animals that
were pre-injected with the unlabeled agent. Specificity of
Cy5.5-labeled EGF was demonstrated in this way by Ke
et al. [26]. Even if differential signal is not readily detected
in intact animals, imaging analysis of excised whole and
sectioned tumors may reveal the competition.
We used this approach to assess specificity of IRDye 800CW
EGF imaging in prostate tumors [27]. Mice bearing either
PC3M-LN4 subcutaneous or orthotopic tumors were injected with IRDye 800CW EGF; some animals were preinjected with C225 anti-EGFR monoclonal antibody. When
we injected tumor-bearing animals with the probe and subsequently performed fluorescence imaging of tumor sections, IRDye 800CW EGF was clearly visible not only in the
primary tumors, but also in lymph nodes extracted from
the animals (Fig. 9). Tumor-bearing animals that were preinjected with C225 exhibited a 33-49% decrease in fluorescent signal, indicating that binding of the labeled ligand
was specific for EGFR.
An alternative approach to demonstrating specific signal is
to quantify targeting agent uptake by both tumor and nontumor tissue. For examining integrin-binding agents,
Becker et al. [53] used an RGD peptide doubly labeled with
125I and an indocarbocyanine dye. Radioactive content of
the tumor, heart, liver, kidneys, and brain was quantified
following imaging of the tumor. Thus, the authors were
able to express the uptake of the probe as the percentage
of injected dose per gram of tissue. This approach may also
be useful for characterization of metabolic probes, for
which high doses of unlabeled competitor may be toxic.
Figure 9. Demonstration of specific tumor targeting
in vivo for the IRDye 800CW EGF optical imaging
agent. Tumors and lymph nodes were excised from
animals bearing either subcutaneous (A) or orthotopic
(B) prostate tumors, as indicated, injected intravenously with IRDye 800CW EGF or pre-injected with
C225 anti-EGFR monoclonal antibody prior to dosing
with IRDye 800CW EGF. The corresponding table presents IRDye 800CW EGF fluorescence signal per area
tissue for vehicle control, optical agent only, and C225
competition for each tumor type (subcutaneous and
orthotopic) and lymph nodes from the orthotopic
tumor model (images in panel C).
Summary and Conclusions
Fluorochrome-labeled molecular probes are valuable tools
for non-invasive longitudinal study of tumorigenesis and
metastasis, preclinical studies of the effects of therapeutic
agents, and pharmacokinetic and pharmacodynamic studies of drug-target interactions. Because of this, these
probes have significant potential for translation to human
clinical use.
35
Page10 – A systematic approach to the development of fluorescent contrast agents for optical imaging of mouse cancer models
Several applications may expand the clinical utility of fluorochrome-labeled probes. For example, accurate definition
of tumor margins is crucial to the therapeutic outcome of
many surgical oncology procedures. A multimodal imaging agent consisting of a magnetic iron oxide nanoparticle
and Cy5.5 has been used as a preoperative nuclear magnetic resonance contrast agent and intraoperative optical
probe to define the tumor margins in a rat gliosarcoma
model [61]. Similar intraoperative imaging technology is
being developed in the laboratory of Frangioni [62]. These
procedures would allow a surgeon to identify and locate
the tumor mass by MRI and subsequently remove the
tumor accurately with visual guidance from an intraoperative near infrared fluorescent imager.
Photodynamic therapy (PDT) is an application that has
been used in oncology for over two decades [9]. A photosensitizing agent delivered to malignant tissue is exposed
to light, generating cytotoxic singlet oxygen. The result is
cell death through the induction of apoptosis, microvascular damage and antitumor immune response. The major
class of dyes used for this approach is phthalocyanines
such as IRDye 700DX [8]. NIR dyes conjugated to antitumor therapeutics such as Erbitux (anti-EGFR monoclonal
antibody), may have similar clinical appeal for simultaneous treatment and monitoring of anti-cancer therapy [63].
Imaging based on expression of luciferase or a fluorescent
protein such as GFP have facilitated examination of intracellular signaling events in vivo. Hybrid gene constructs in
which either the luciferase or GFP gene is placed under
control of an inducible promoter responsive to a signaling
pathway of interest have been used to directly assess the
effects of anti-tumor agents on the gene regulation in vivo.
A similar reporter system that directly images
β-galactosidase activity on a far red substrate has been recently reported ([64]. This fluorescent reporter system
could provide a means for creating hybrid expression units
to examine in vivo gene expression and regulation of a
number of important pathways in cancer.
NIR-based imaging instrument technologies designed for
human clinical use are in various stages of development
[62, 65, 66]. Combined with new NIR-labeled biomarkers,
these will expand the clinical options available for cancer
management in the near future. Several excellent reviews
describe the use of targeted markers in animal studies
[7, 67].
In this review we have discussed the basic validation of fluorochrome-labeled molecular probes. Although near-infrared optical excitation sources provide deeper tissue
penetration, they are not great enough to provide unlimited application for human clinical use. Near-infrared imaging agents may be limited to accessible tissues such as
breast tumors, or to intraoperative applications, endoscopy, and photodynamic therapy. However, these applications represent significant benefits for research,
diagnosis, and treatment. Continued technological innovation in imaging instrumentation, biomarker discovery,
36
PUBBLICAZIONI
and labeling chemistries will foster the clinical use of fluorescent optical imaging.
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38
PUBBLICAZIONI
Proteomics 2007, 7, 1753–1756
1753
DOI 10.1002/pmic.200601007
TECHNICAL BRIEF
Differential protein labeling with thiol-reactive infrared
DY-680 and DY-780 maleimides and analysis by
two-dimensional gel electrophoresis
Irène M. Riederer1 and Beat M. Riederer1, 2
1
2
Centre de Neurosciences Psychiatriques, Hôpital Psychiatrique, Prilly, Switzerland
Département de Biologie Cellulaire et de Morphologie, Université de Lausanne, Lausanne, Switzerland
Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities.
Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to
maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure
allows amounts of less than 5 mg of cysteine-labeled protein mixtures to be detected (together
with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains
are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins,
such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer
detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides
an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.
Received: December 6, 2006
Revised: January 30, 2007
Accepted: March 2, 2007
Keywords:
Immunoblots / Infrared dyes / Mouse brain / Thiol labeling
Applications that include protein labeling with sensitive
fluorescent dyes such as SYPRO Ruby and SYPRO Orange
or cyanine dyes (Cy2, Cy3, and Cy5) have been widely used in
DIGE for the detection of differences in the protein composition of various sets of proteins [1]. Here, we describe the
use of infrared DY-680 and DY-780 maleimides to label two
sets of proteins for rapid identification of differences in their
protein composition by 2-DE. The method is reproducible
and dyes can be obtained at a reasonable price. One drawback is that only cysteine-containing proteins are labeled,
Correspondence: Dr. Beat M. Riederer, Centre de Neurosciences
Psychiatriques, Hôpital Psychiatrique, 1008 Prilly, Switzerland
E-mail: [email protected]
Fax: 141-21-692-51-05
Abbreviations: Cy, cyanine dyes; GFAP, glial fibrillary acidic protein; SCG10, stellar cervical ganglion protein 10
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
thus excluding proteins without cysteines. As an example,
newborn and adult mouse brain proteins were labeled with
DY680- and DY780-maleimides (outlined in Fig. 1). These
stains have an Mr of 756.97 g/mol for DY-680 (C42H52N4O7S)
and 783.01 g/mol for DY-780 (C44H54N4O7S), a neutral pH,
and differ in their infrared light emissions by 100 nm due to
a difference in a vinyl residue (www.dyomics.com). Preliminary experiments with individual DY-680 or DY-780-labeled
proteins separated on 1-D gels showed no overlap in emission with the measuring wavelengths of 800 and 700 nm,
respectively. It should be noted that the maleimide dyes are
also available as NHS-conjugated dyes that could be applied
for labeling of all proteins via primary and e-amino groups;
such experiments are currently being performed.
All experiments were authorized by the local veterinary
office. Brains from deeply anesthetized newborn and adult
mice (C57Bl/6) were removed and kept at –807C until use.
Brain samples (25 mg brain tissue or 2.5 mg proteins) were
www.proteomics-journal.com
39
1754
I. M. Riederer and B. M. Riederer
Figure 1. Summary of experimental set-up and procedure. Panel
A represents the common separation of the two differentially
labeled protein samples in 2-DE IEF pH 3–10 in the first dimension and by molecular weights in the second dimension (panel
A); and detection for wavelength specific emission at 700 nm
(newborn mouse brain, panel B) and 800 nm for adult brain proteins (panel C). For comparison unlabeled newborn (panel D) and
adult (panel E) CBB-stained brain proteins are shown for comparison. Arrows point to two stage-specific proteins, GFAP in
green and SCG10 in red (panel A). The molecular weights are
indicated to the right in panel A.
homogenized in 2.5 mL reducing buffer (0.1 M sodium
phosphate pH 6.0, 2.5 mM EDTA, 5 mL/mL protease inhibitors (Sigma, Buchs, Switzerland) and 6 mg 2-mercaptoaethylamine-HCL) and incubated for 90 min at 377C. In
addition, two samples of purified pig brain tubulin (0.5 mg)
[2] were labeled with each dye for a dilution series and for an
estimation of the LOD of a known protein. The reducing
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
40
PUBBLICAZIONI
Proteomics 2007, 7, 1753–1756
buffer was replaced by a D-salting Dextran desalting column
(Perbio Switzerland, Lausanne), previously equilibrated with
conjugation buffer (PBS, pH 7.5 and 1 mM EDTA). Subsequently 500-mL fractions were collected and protein amounts
were measured by the Bradford Protein Assay (BioRad,
Reinach, Switzerland) or the dot assay and by staining filters
with Amidoblack [2]. Protein-containing fractions were
pooled and concentrated to 500 mL by a centrifugal filter unit
(Amicon Ultra-4, Millipore, Zug, Switzerland). Infrared substances DY-680 and DY-780 maleimides were obtained from
Dyomics (Jena, Germany). Two hundred micrograms of DY680 maleimide in 20 mL DMF was added to 2.5 mg brain
proteins of newborn mice and 200 mg DY-780 maleimides in
20 mL DMF was added to adult brain protein samples and
kept overnight at 47C for saturation labeling of all cysteines.
Subsequently, 40-mg dye samples in 4 mL DMF were added to
tubulin samples and also kept over night at 47C. Samples
were desalted with D-salt Dextran columns and concentrated
by Amicon Ultra-4 centrifugation. A considerable amount of
proteins are lost during desalting and concentration procedures, but between 25 and 40% of the starting material is
obtained in dye-labeled form. For 2-DE, both labeled and
unlabeled proteins were treated with the 2-D clean-up kit
(Amersham, Otelfingen, Switzerland) and resuspended in
strip-rehydration buffer for IEF or in SDS-PAGE sample
buffer. Procedures for 1- and 2-DE as well as for silver nitrate
and CBB staining were previously described [3–5]. Briefly,
5 mg labeled proteins and 400 mg unlabeled proteins were
loaded on IPG ready strips of 11 cm length and pH 3–10
(BioRad) in the first dimension; in the second dimension,
12.5% or gradient 3.6–15% SDS-PAGE was applied. Gels
were scanned by the Odyssey infrared imaging system (LiCor, Bad Homburg, Germany) with detection channels of
700 nm (red) and 800 nm (green) at a sensitivity level setting
of 5, a scanning resolution of 169 mm, and at medium quality. Data sets were analyzed by computer software (Image
master, Amersham/GE Biosciences, Zurich). Proteins were
also transferred to NC filters and Western blots were
exposed to antibodies for tubulin, glial fibrillary acidic protein (GFAP), and neurofilament proteins [4, 5] and detected
with infrared-conjugated secondary antibodies IRD-700DX
or IRD800DX (Rockland, BioConcept, Allschwil, Switzerland). The minimal protein amount that can be used for
labeling is 200 mg proteins as starting material, yielding 40–
50-mg labeled proteins. This is sufficient for several 2-D gels,
while the same amount of unlabeled proteins is only sufficient for a single 2-D gel, CBB staining, and identification
of proteins.
The sample preparation and labeling process of adult
and newborn mouse brain proteins is demonstrated in
Fig. 1. The newborn DY-680-labeled proteins appear in red,
while the adult DY-780-labeled proteins (5 mg each) are seen
in green (Fig. 1A). The two emission patterns were transformed into black and white images, with newborn protein
composition represented in panel B and the adult protein
pattern in panel C. For comparison, unlabeled newborn and
www.proteomics-journal.com
Proteomics 2007, 7, 1753–1756
adult brain proteins (400 mg each) were separated individually and stained with CBB (ProtoBlue Safe, National Diagnostics, Chemie Brunschwig, Basel, Switzerland). The CBBstained proteins are quite useful for MALDI-TOF identification and for comparison with DY-labeled protein patterns.
Despite a certain similarity, it is apparent that a variety of
proteins are not present in the dye-labeled sample. Several
proteins do not contain cysteine residues [6] and are therefore not labeled, escaping detection in the infrared scanner.
Furthermore, there are development-related differences in
protein pattern, e.g. the GFAP (Fig. 1A, arrow pointing to
green protein) [5] is more prominent in adult brain while
stellar cervical ganglion protein 10 (SCG10; arrow pointing
to red spots) is more present in newborn brain. The amino
acid composition of these two proteins reveals a single cysteine residue for GFAP (Swiss-Prot no. P03995) and two
cysteines for SCG10 (P55821). Therefore, a successful labeling of proteins with only a minimal number of cysteines per
molecule is possible.
In Fig. 2, a dilution series of labeled tubulin demonstrates that the detection of labeled brain tubulin is possible
down to 1 fg (Fig. 2A). Given that tubulins contain seven to
eight cysteines, the sensitivity limit of the method is approximately 10 fg. A separation on 1-DE already demonstrated
that differences in protein labeling may occur (Fig. 2B). On
Western blots, a mAb for tubulin (Tu9b) clearly stained btubulin in the unlabeled adult brain protein sample (Fig. 2 C,
lane 2), while in the DY780-labeled sample no positive
detection was possible and staining appeared rather as a
nonspecific smear. A similar result was obtained with two
antibodies for NF-L and GFAP. This suggests that covalently
bound maleimide dyes may interfere with immunological
detection or alter epitopes. It remains to be seen whether
proteins that do not contain cysteines are still immunologically detectable. Regarding the influence of dyes on MALDITOF protein identification, corresponding and equal
amounts of samples from the tubulin region were cut out of
CBB-stained DY-labeled and non-DY labeled samples
(http://www.dyomics.com/48.html) [7] and tested for their
suitability to MALDI-TOF analysis by the Protein Analysis
Facility (PAF) in Epalinges, Switzerland. Tubulins a-1, b-2, b3, and b-5 were successfully identified in both samples with
MASCOT scores for unlabeled tubulins between 899 and
1017 and for DY-780 labeled tubulin between 285 and 677
(http://www.dyomics.com/48.html) [7]. This indicates that
tubulin identification is still possible, but given the lower
scores for dye-labeled proteins, one must suspect that thiollabeled peptide sequences may no longer allow identification
of the correct peptide mass. Tubulins contain between seven
and eight cysteines, and therefore several peptides may
become unavailable, thus explaining the lower MASCOT
scores.
Protein labeling with DY-680 and DY-782 maleimides
was shown to be a sensitive method for detecting minute
quantities of proteins by 2-DE and infrared laser scanning,
with a sensitivity in the femtogram range. The physical
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Technology
1755
Figure 2. The LOD of IR680-labeled tubulin is in the femtogram
range (A), the blank areas (asterisk) represent a saturated protein
concentration. One-dimensional SDS-PAGE with newborn (nb),
adult proteins (ad) or combined (mix) demonstrates that a 1-D
representation is by far sufficient to identify differences (B), the
molecular weights are indicated to the right. Panel C: An infraredlabeling of cysteines altered immunoreactivity of labeled (1) but
not unlabeled (2) tubulin. Note that the immunoreactivity
appears like a smear in lane 1 and not as specific reactivity with
tubulin in lane 2.
properties of infrared dyes did not alter the migration properties very much in the 2-DE separation, since the charge and
size of many proteins remained similar or the same. The
dye-labeled GFAP and SCG10 proteins are found more or
less at the same places as identified in the CBB-stained gel.
Proteins without cysteine residues are obviously not labeled
and therefore escape detection in the Odyssey infrared imaging system.
The high sensitivity of the detection system allows the
use of only a few micrograms of proteins per 2-D gel, but the
protein amounts of most protein spots in such a 2-D gel are
far from sufficient for a positive MALDI-TOF identification,
requiring either higher amounts of DY-labeled proteins or
the addition of unlabeled proteins and a CBB or silver staining for the identification of matching proteins by MS. It has
been shown that DY-680- and DY-780-labeled proteins can
also be stained with CBB and silver nitrate [7], but it is
recommended to image proteins first with an infrared scanner, since CBB-stained and silver nitrate-stained proteins
interfere with the infrared imaging. Since detection with
CBB and silver nitrate staining is possible, other noncovalent
www.proteomics-journal.com
41
1756
I. M. Riederer and B. M. Riederer
stains such as SYPRO Ruby, Deep Purple or Lightning Fast
may also work for the detection of DY-labeled proteins. It
should also be noted that on Western blot filters the proteins
are brought to the same focal plane. This favors a better
detection by the infrared imaging system and may also allow
detection with a variety of noncovalent protein stains [1, 2].
Infrared labeling and DIGE provides an additional, inexpensive tool for identifying differences in cysteine-containing
proteins between two developmental stages, two experimental conditions, or between a control and a pathological
state [2]. By using five or more dyes and an imaging system
with appropriate filters, the separation of many samples
within a single 2-D gel seems feasible.
In the present protocol, all thiol groups were reduced and
the oxidation-sensitive modifications were lost and no longer
distinguishable by 2-D DIGE. It is known that protein oxidation plays a crucial role during aging and a variety of diseases [8]. Therefore, the labeling protocol could be modified
so that a given sample is labeled with one color prior to the
reduction of oxidized cysteines, followed by reduction of oxidized proteins and labeling of the reduced cysteines with
another color [2]. The identification of oxidized proteins may
find a wide application in the study of different pathologies
and during normal aging.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
42
PUBBLICAZIONI
Proteomics 2007, 7, 1753–1756
The authors thank Dr. F. Lehmann (Dyomics GmBH) for
his helpful suggestions and support. This work was supported by
an FNRS grant (31-067201.01).
References
[1] Patton, W. F., J. Chromatogr. B: Anal. Technol. Biomed. Life
Sci. 2002, 771, 3–31.
[2] Bondallaz, P., Barbier, A., Soehrman, S. et al., Cell Motility
Cytoskeleton 2006, 63, 681–695.
[3] Porchet, R., Probst, A., Bouras, C. et al., Proteomics 2003, 3,
1476–1485.
[4] Porchet, R., Probst, A., Dráberova, E. et al., Neuroreport 2003,
14, 929–933.
[5] Shaw, M., Riederer, B. M., Proteomics 2003, 3, 1408–1417.
[6] Patton, W., Electrophoresis, 2000, 21, 1123–1144.
[7] Riederer, I. M., Riederer, B. M., in: Palagi, P. M., Quadroni, M.,
Rossier, J. S., Sanchez, J. C., Stöcklin, R. (Eds.), Proceedings
of the Swiss Proteomics Society, Fontis Media, Bern, Switzerland, 2004, pp. 183–185.
[8] Paget, M. S. B., Buttner, M. J., Annu. Rev. Genet. 2003, 37, 91–
121.
www.proteomics-journal.com
PUBBLICAZIONI ODYSSEY
Substitution of the use of radioactivity by
fluorescence for biochemical studies of RNA
Bei-Wen Ying, Dominque Forumy and Satoko Yoshizawa
Laboratoire de Chimie et Biologie Structurales, ICSN-CNRS, 91190 Gif-sur-Yvette, France
Abstract
We present here the use of fluorescent methodologies for structural and functional studies of
RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of
an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases
of RNA. The new fluorescent primer extension methodology allows simple and fast chemical
probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used
to monitor protein–RNA interactions by fluorescent mobility shift assays. The speed and ease
of these approaches are advantages over prior methods that used hazardous radioisotopes.
Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.
43
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TRIS-GLYCINE DRY PACK
FE3 40016B
TRIS-GLYCINE SDS DRY PACK
FE3 340030
NEWBLOT NITRO STRIPPING BUFFER
FE3 340032
NEWBLOT PVDF STRIPPING BUFFER
FE3 340040
SMARTGEL 7,5% 100 ml
FE3 340040
SMARTGEL 7,5% 500 ml
FE3 340040
SMARTGEL 10% 100 ml
FE3 340040
SMARTGEL 10% 500 ml
FE3 340040
SMARTGEL 12,5% 100 ml
FE3 340040
SMARTGEL 12,5% 500 ml
IN-CELL WESTERN
FE3 310700
ODYSSEY ICW KIT 1
FE3 310720
ODYSSEY ICW KIT 2
kit per ICW contenente:
IRDye 800 CW Goat Anti-Mouse secondary antibody (0.5 mg)
Odyssey Blocking Buffer (4x500 ml)
Draq5 TM (100 ml)
Sapphire700 TM Stain (100 ml)
(reagenti per 40 piastre da 96 pozzetti o 10 piastre da 384 pozzetti)
kit per ICW contenente:
IRDye 800 CW Goat Anti-Rabbit secondary antibody (0.5 mg)
Odyssey Blocking Buffer (4x500 ml)
Draq5 TM (100 ml)
Sapphire700 TM Stain (100 ml)
(reagenti per 40 piastre da 96 pozzetti o 10 piastre da 384 pozzetti)
STAINING E MARCATURA DI PROTEINE ED ANTICORPI
FE3 0928040
HIGH MOLECULAR WEIGHT IRDye 800 CW PROTEIN
LABELLING KIT
Kit per la marcatura di 3x1.0 mg di proteina
Adatto per proteine da 45 a 194 kDa
FE3 0928042
LOW MOLECULAR WEIGHT IRDye 800CW PROTEIN
LABELLING KIT
Kit per la marcatura di 3x1.0 mg di proteina
Adatto per proteine da 10 a 45 kDa
57
Overlay
700 nm (normalization)
800 nm (pERK)
Total DRAQ5/
ERK Sapphire700
Total DRAQ5/
ERK Sapphire700
Total DRAQ5/
ERK Sapphire700
Metodo di Normalizzazione
0 .9
Background
pERK/ERK
pERK/DRAQ5+Sapphire
0 .8
0.8
0 .6
0 .3
0
24
Rel. Flluorescence Intensity
800 Ch Integrated Intensity
1 .2
18
Fold-activation of ERK
pERK/ERK
pERK/DRAQ5+Sapphire
1 8.3
12
6
0
Confronto dei Metodi di Normalizzazione
L’attivazione di ERK è stata indotta in cellule A431 stimolando con EGF. Fosfo-ERK è
stato misurato ad 800 nm usando un anticorpo anti fosfo-ERK e un secondario marcato
con IRDye800. la normalizzazione è stata eseguita in due diversi modi: quantificando
la quantita di ERK totale con un anticorpo anti ERK ed un secondario marcato con
IRDye 680, oppure con colorazione con DRAQ5 e Sapphire700. Entrambi i metodi
garantisco lo stesso risultato di normalizzazione dell’espressione di fosfo-ERK.
58
PRODOTTI PER IMAGING INFRAROSSO
19 .9
FE3 0928044
MICROSCALE MOLECULAR WEIGHT IRDye 800CW PROTEIN
LABELLING KIT
Kit per la marcatura di 3x1.0 mg di proteina
Adatto per proteine da 10 a 194 kDa
FE3 338046
HIGH MOLECULAR WEIGHT IRDye 700DX PROTEIN
LABELLING KIT
Kit per la marcatura di 3x1.0 mg di proteina
Adatto per proteine da 45 a 194 kDa
FE3 338048
LOW MOLECULAR WEIGHT IRDye 700DX PROTEIN
LABELLING KIT
Kit per la marcatura di 3x1.0 mg di proteina
Adatto per proteine da 10 a 45 kDa
FE3 338050
MICROSCALE MOLECULAR WEIGHT IRDye 700DX PROTEIN
LABELLING KIT
Kit per la marcatura di 3x1.0 mg di proteina
Adatto per proteine da 10 a 194 kDa
FE3 400020
IRDye BLU COOMASSIE, 1 litro
Blu Coomassie per lo Staining dele proteine totali
FE3 400220
SAPPHIRE TM 700 STAIN, 100 μl
per staining di normalizzazione dei ICW
FE3 700100
IRDye 700DX INFRARED Dye NHS ESTER (0,5 mg, liofilizzati)
FE3 700110
IRDye 700DX INFRARED Dye NHS ESTER (5,0 mg, liofilizzati)
FE3 700200
IRDye 800CW INFRARED Dye NHS ESTER (0,5 mg, liofilizzati)
FE3 700210
IRDye 800CW INFRARED Dye NHS ESTER (5,0 mg, liofilizzati)
FE3 720200
IRDye 800RS INFRARED Dye NHS ESTER (0,5 mg, liofilizzati)
FE3 720210
IRDye 800RS INFRARED Dye NHS ESTER (5,0 mg, liofilizzati)
IRDye® 700DX in PBS
100
100
ACQUA
1XPBS
210,000
165,000
165,000
680
689
689
EM
MAX
(nm)
687
700
700
MW
(g/mole)
1954
1954
1954
90
IRDye 700DX
90
Emission
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
550
600
650
700
750
0
800
Wavelength (nm)
59
Relative Fluorescence
METANOLO
ABS
MAX
(nm)
Relative Absorbance
DILUENTE
EXT.
COEFF.
(M -1 cm -1)
Absorbance
300,000
240,000
ACQUA
240,000
1XPBS
PBS: METANOLO
270,000
ABS
MAX
(nm)
778
774
774
777
EM
MAX
(nm)
794
789
789
791
IRDye® 800CW in PBS
MW
(g/mole)
Absorbance
90
1166
1166
1166
1166
100
100
IRDye 800CW
90
Emission
80
80
70
70
60
60
50
50
40
40
30
30
20
20
Relative Fluorescence
METANOLO
EXT.
COEFF.
(M -1 cm -1)
Relative Absorbance
DILUENTE
10
10
0
600
650
700
750
800
850
0
900
Wavelength (nm)
300,000
200,000
ACQUA
200,000
1XPBS
PBS: METANOLO
270,000
ABS
MAX
(nm)
770
767
767
770
EM
MAX
(nm)
786
786
786
786
IRDye® 800RS in 1x PBS
MW
(g/mole)
100
100
Absorbance
90
90
Emission
961
961
961
961
IRDye 800RS
80
80
70
70
60
60
50
50
40
40
30
30
20
20
10
10
0
600
650
700
750
Wavelength (nm)
IN-VIVO IMAGING
FE3 0926446
IRDye 800CW EGF OPTICAL PROBE
Recombinant EGF polypeptide, contenente 54 residui
aminoacidici coniugati con il fluoroforo IRDye 800CW
Due topi atimici, con tumore, iniettati
con 0,9% di soluzione salina (sinistra) e
con 1 nmol di EGF marcato con IRDye
800 (destra), 72 ore prima dell’imaging
su Odyssey e Mouse-POD.
60
PRODOTTI PER IMAGING INFRAROSSO
800
0
850
Relative Fluorescence
METANOLO
EXT.
COEFF.
(M -1 cm -1)
Relative Absorbance
DILUENTE
NORTHERN BLOT, SOUTHERN BLOT ED EMSA
FE3 322310
IRDye 680 LABELED STREPTAVIDIN, 0,5 mg
FE3 322300
IRDYE 800CW STREPTAVIDIN, 0,5 mg
FE3 079210
OLIGO EMSA PER p53 (IrDye 700), 50 nM
FE3 079220
OLIGO EMSA PER STAT3 (IrDye 700), 50 nM
FE3 079230
OLIGO EMSA PER CREB (IrDye 700), 50 nM
FE3 079240
OLIGO EMSA PER NFKB (IrDye 700), 50 nM
FE3 079250
OLIGO EMSA PER AP-1 (IrDye 700), 50 nM
FE3 079260
OLIGO EMSA PER SP-1 (IrDye 700), 50 nM
FE3 079270
OLIGO EMSA PER E2F (IrDye 700), 50 nM
FE3 079280
OLIGO EMSA PER EGR (IrDye 700), 50 nM
FE3 079290
OLIGO EMSA PER HIF-1 (IrDye 700), 50 nM
FE3 079300
OLIGO EMSA PER OCT-1 (IrDye 700), 50 nM
FE3 079310
OLIGO EMSA PER PAX-5 (IrDye 700), 50 nM
FE3 079320
OLIGO EMSA PER YY1/NFE1 (IrDye 700), 50 nM
Analisi nell’infrarosso dell’EMSA di AP1
AP1-EMSA è stato eseguito per valutare i
cambiamenti del legame di AP1 in tre diversi
trattamenti su cellule HeLa. HeLa controllo,
HeLa a 2 ore (dati non mostrati) e HeLa a
4 ore in risposta dopo stimolazione con
siero. Gli estratti nucleari sono stati caricati
a diluizioni seriali e identificati usando un
oligo marcato con IRDye 700 contenente
la sequenza del AP1 binding domain.
61
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Le Guide
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GUIDA ALL'IMAGING INFRAROSSO
L'analisi quantitativa dell'espressione proteica
PCR QUANTITATIVA
Guida Metodologica ed Applicativa
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