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