Preparazione del campione
DENATURANTE
SOLUBILISATION
and
or
EXTRACTION
Different components
8 M Urea
o
7 M Urea and 2 M Thiourea
1 - 4 % Surfactants [w/v]
0.5 - 1 % Ampholine [w/v]
Reducing treatment
Zwitterionic
CHAPS
CHAPSO
Sulfobetaines
NON-ionic
NP-40
Triton
Tween
Brij
DTE 20 - 100 mM
TBP 5 mM
TCEP 10 mM
Detergente
Sodium dodecyl
sulfate
CMC [mM]
Mr [Da]
Mr micella
[Da]
8
288
18000
Sodium
deoxycholate
1-4
415
4200
CHAPS
8-10
615
6150
CHAPSO
8-10
631
6940
Zwittergent 3-14
0.3
364
30000
Brij 35
0.09
1168
NP-40
0.3
603
Triton X-100
0.3
650
Tween 20
0.059
1228
90000
Struttura
Fluid-mosaic model of membrane structure
2% ABS 14
2% TWEEN 40
2% a Dodecyl Maltoside
2% b Dodecyl Maltoside
2% Plantacare
2% Brij 30
2% Brij 56
2% Brij 58
2% Brij 78
2% Brij 96
1% Triton X-100
4% CHAPS
Sylvie Luke et al. Proteomics 2003, 3, 249-253.
Sequential detergent extraction
of membrane proteins of Human RBC cells
b-Hexyl-D-glucopiranoside
Triton X-100
&
Deoxycholate
Pellet
Reduction and Alkylation reaction
H+
S-S
S-S
H
TBP+ O-
S-
HI
NH2
I
S
O
SH
-S
TBP =O
NH2
=
H2O
=
TBP
or
DTT - DTE
2 H+
O
Reduction, NO Alkylation
6
NL pH
Reduction AND Alkylation
11
NL pH
7
KDa
KDa
250
100
250
100
10
50
37
50
25
15
10
37
25
15
Carbonic anhydrase
Thioredoxin peroxidase
Biliverdin reductase B
In the absence of alkylation, homo- and hetero-oligomers are
formed, up to dodecamers!
Spot 1
b
Spot 3
a
Spot 5
b-b
Spot 8
a-a
Spot 13
a-b
Elettroforesi bidimensionale (2-DE)
Vantaggi condizioni denaturanti IEF
IEF in presenza di 8-9 mol/L urea, detergenti non-ionici, riducenti
• proteine in un’unica conformazione
• urea e detergenti:
- evitano aggregazioni proteiche
- mantengono le proteine idrofobiche in soluzione
• si evitano differenti step di ossidazione
• pI teorici molto simili a pI in condizioni denaturanti
Rimozione di fattori che disturbano
la separazione
•
•
•
•
•
•
•
•
micro-dialysis (too long)
TCA precipitation (poor efficiency)
Acetone precipitation (good)
precipitation with TCA in acetone (poor
efficiency)
precipitation with methanol-chloroform (only for
small volumes)
precipitation with tributyl phosphate-acetonemethanol
other methods
commercial tools
Microdialisi
Lymphocyte pellet
Thermal shock
Urea 7M Thiourea 2M CHAPS 4%
DNase RNase
First dimension on
3-10 pH gradient
200 μg protein
Dialysis
Tributylphosphine
Ampholyte 3-10 non linear
Albumin depletion
Piastrine
3
NL pH
3
10
200
Platelets
1995
NL pH
10
non muscle
myosin
Platelets now
Mr
kDa
Gravel P, et al
Electrophoresis
16:1152-1159, 1995
Platelets reduced & alkylated prior to
the IPG run + precipitated in acetone,
methanol, tributyl phosphate
Bruschi M, Musante L, Candiano G, Ghiggeri GM,
Herbert B, Antonucci F, Righetti PG:
Soft IPG gels in proteome analysis: a follow-up.
Proteomics 2003 3 821-5
5
Elettroforesi bidimensionale (2-DE)
Visualizzazione degli spots proteici
Limiti di Sensibilità
Quantificazione
Comassie blue
100 ng
+++
Silver staining
200 pg
++
Fluorescent staining
10 – 100 pg
++++
Radioactive labeling
1 pg
+++
Analisi delle immagini
•Image scanner (trasmissione e
riflessione)
• Fluorimager (nile red; sypro orange;
sypro Ruby)
• Imagemaster (per piccoli gel)
Image Scanner
• Comparazione tra cellule di
controllo e cellule trattate;
• sovrapposizione di immagini
con colori diversi
• individuazione e quantificazione
degli spots proteici mediante
comparazione con le banche dati
Metodi di colorazione del gel
Stain
Limit
of
detection
Linear
dynamyc
range
Compatible
with Mass
Spectrometry
Acid Silver
1-2 ng
8-60 ng
No-Yes
Alkaline Silver
1-2 ng
8-60 ng
No
Colloidal
5-10 ng
10-250 ng
Yes
Coomassie
50-100 ng
150-1000 ng
Yes
DIGE ®
Fluorescence 2D difference gel electrophoresis: DIGE
Protein extract 1
Labeled with fluor 1
Mix labelled extracts
Protein extract 2
Labeled with fluor 2
2D-PAGE
Image gel
Excitation
Wavelength 2
Excitation
Wavelength 1
Analysis of difference
Image analysis:
Overlay images
Image analysis:
Data quantitation
Comparison of “Secretoma” from Normal and Pharmacological Treatment
of bronchial epithelial cells
Control [Cy5]
Pharmacological Treatment [Cy3]