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 CO2incubator 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.

·  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

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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