Molecular and Clinical Radiobiology Workshop Lab Practical Tutorial Sessions RI-MUHC
June 19th, 2015
PROTOCOL
Analysis of H2AX phosphorylation by Western Blot
Cell culture and treatment
Culture the cells on 100mm cell culture dishes, according to the requirement (modified
Eagle’s MEM supplemented with 10% fetal bovine serum (FBS), vitamins, sodium
pyruvate, L-glutamine, penicillin, streptomycin, and nonessential amino acids at 37°C in
the presence of 5% CO2).
Treat the cells as necessary with radiation (e.g., 2-10 Gy)
Incubate the cells in a CO2 incubator at 37 °C for 0 min, 5min, 30min, 60min, 6hs and
24hs.
Protein extraction and quantification
Wash cells with cold PBS and then lyse with ice cold RIPA buffer for 45 minutes at 4°C.
Centrifuge the extracts at 12000g for 15minutes at 4°C. Collect the supernatant
containing total extracted proteins. BCA protein assay kit (Pierce Scientific Ltd) will be
used for protein quantification.
SDS-PAGE and Western blot
Load samples (30-50ug protein) on a polyacrylamid gel (4.0% stacking gel and 15%
running gel).
Run gel at 100 V (Bio-Rad Mini-Protean gel electrophoresis system will be used).
Transfer proteins from the gel to the membrane
Equilibrate gel in transfer buffer for 15 minutes.
During equilibration, cut nitrocellulose to the size of the gel; wet in water and soak in
transfer buffer.
For each gel, cut 2 pieces of 3 MM paper to size of gel and soak in transfer buffer.
Wet pads with transfer buffer.
Assemble transfer unit as outlined below:
(Red) pos. pole> clear plate> pad> 3 MM> nitrocellulose> gel> 3 MM> pad> black
plate> neg. pole (Black). Make sure no bubbles are trapped
Close the sandwich board and dunk into partially filled transfer chamber.
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Molecular and Clinical Radiobiology Workshop Lab Practical Tutorial Sessions RI-MUHC
June 19th, 2015
Put in "BioIce" and fill chamber to top, but do not over fill.
Run transfer at 30 volts overnight in a cold room .
Verify transfer by staining with Ponceau S dye
Block membrane for 1 h at room temperature on an orbital shaker using 5% (w/v) non fat
milk in TBST.
Incubate with primary antibody diluted in TBST (1:1000 - Phospho-Histone H2A.X
(Ser139) (20E3) Rabbit mAb #9718 Cell Signaling) overnight at 4 °C on an orbital
shaker.
Pour off primary antibody solution and wash 3 x 10 min in TBST at room temperature.
Incubate with secondary antibody (Anti-rabbit IgG, HRP-linked Antibody #7074 - Cell
Signalling) diluted (1:2,000) in 5% milk in TBST for 1 hr at room temperature on an
orbital shaker.
Pour off secondary antibody solution and wash 3 x 10 min in TBST at room temperature.
ECL chemi-luminescence [Amersham Biosciences (GE)] will be used to detect protein
bands. Manual band quantification will be carried out using Spectrum Image and
GelQuant® software.
Reagents
Lysis buffer (RIPA)
10 mM Tris-Cl (pH 8.0)
1 mM EDTA
0.5 mM EGTA
1% Triton X-100
0.1% sodium deoxycholate
0.1% SDS
140 mM NaCl
1 mM PMSF
Add Protease and Phosphatase inhibitors (Roche 04693132001 and 04906845001)
Protein sample buffer
40 mM Tris-HCl, pH 6.8
8 M Urea 5% (w/v) SDS
0.1 mM EDTA
1% (v/v) β-Mercaptoethanol (freshly added)
0.1 g/L Bromphenolblue
Running buffer
25 mM Tris
192 mM Glycine
0.1% (w/v) SDS
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Molecular and Clinical Radiobiology Workshop Lab Practical Tutorial Sessions RI-MUHC
Transfer buffer
25 mM Tris
192 mM Glycine
20% (v/v) Methanol
Check the pH and adjust to pH 8.3 if necessary
Ponsceau S - store at RT
0.1% w/v Ponsceau S
in 1% v/v acetic acid
Tris-buffered saline (TBS )
25 mM Tris
140 mM NaCl
2.5 mM KCl
pH 7.4
Tris-buffered saline with Tween 20 (TBST)
TBS with 0.1% (v/v) Tween 20
Phosphate buffered saline (PBS)
140 mM NaCl
2.5 mM KCl
8.1 mM Na2HPO4
1.5 mM KH2PO4
pH 7.3
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June 19th, 2015
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