1. Preparation of whole cell lysates from adherent 293 cell culture
1.1. Pre-cool RIPA lysis buffer on ice. Add PMSF (100x), NaF (100x), Na3VO4(250x) and protease inhibitor (25x) to final 1x.
1.2. Place cell culture on ice and wash the cells with ice-cold PBS.
1.3. Drain the PBS, then add ice-cold lysis buffer (1ml per 107 cells, ~ 0.7 ml for 10 cm dish, ~1.8 ml for 15 cm dish).
1.4. Scrape adherent cells off the dish using a plastic cell scraper. Gently transfer the cell suspension into a pre-cooled microfuge tube.
1.5. Leave cell suspension on ice for 30 min with occasional mixing.
1.6. Centrifuge at 10,000 x g for 20 minutes at 4C.
1.7. Gently remove the tube from the centrifuge and place on ice. Carefully collect the supernatant, without disturbing the pellet and transfer to a new clean tube and discard pellet.
1.8. The protein concentration can be determined by Bradford or BCA assay (1-5 mg/ml).
1.9. The cell lysate can be frozen at this point for long-term storage at -80C.
2. Immunoprecipitation
2.1. Thaw lysate on ice. Take 1000 ug whole cell lysate, add 2 ug of immunoprecipitation antibody. Add cold PBS with all inhibitors to make final volume 1ml.
2.2. Incubate 2 h at 4C on a tube rotator or orbital shaker.
2.3. Resuspend the immobilized protein A agarose bead slurry (Thermo Fisher Cat# 20333) by gently vortexing. Remove 50ul and wash in cold PBS. Resuspend in 50 ul cold PBS with all inhibitors. Add the bead slurry to lysate.
2.4. Incubate overnight at 4C on a tube rotator or orbital shaker.
2.5. Centrifuge the tube at 2,500xg for 30 seconds at 4C.
2.6. Carefully remove supernatant completely and wash the beads 2 times with 500 ul of cold PBS w/PMSF, centrifuge to pellet beads in between washes. In order to minimize background, care should be given to remove the supernatant completely following each wash.
2.7. After the last wash, carefully aspirate supernatant and add 50 ul of 2X SDS-PAGE sample loading buffer to bead pellet.
2.8. Vortex and heat to 90C for 10 minutes.
2.9. Centrifuge at 10,000xg for 5 minutes, carefully collect supernatant and load 20 ul onto the gel. Alternatively, the supernatant samples can be collected, transferred to a clean tube and frozen at -80C if the gel is to be run at a later stage.
3. SDS-PAGE and Immunoblotting (Western Blotting, WB)
3.1. Run SDS-PAGE at 180V for 60 min.
3.2. Transfer proteins from the gel onto PVDF or nitrocellulose membrane at 100V for 60 min.
3.3. Place the membrane into the blocking buffer (5% non-fat milk in PBST) and incubate for 30 min at room temperature on a rocking platform.
3.4. Prepare the primary immunoblotting antibody in blocking Buffer. Use a standard concentration of 1 ug/ml. Incubate the blot in primary antibody for at least 2 hours at room temperature or overnight at 4C on rocking platform.
3.5. Wash the blot 3 times in TBS-T for 5 minutes each.
3.6. Prepare the secondary goat anti-rabbit IgG antibody (GeneTex GTX221666-01) at a 1:1,000 dilution in PBST. Incubate the blot secondary antibody for 1 hour at room temperature on a rocking platform.
3.7. Wash the blot 3 times in TBS-T for 5 minutes each.
3.8. Develop the blot using the Chemiluminescent HRP substrate following the manufacturer's instructions.
0.1 M PMSF Protease Inhibitor (100X):
Dissolve 0.174 g PMSF (MW 174.2) in 10 ml EtOH. Aliquot and store at -20C. Before use, take out to RT and completely dissolve PMSF crystals.
Protease Inhibitor Cocktail (25X)
Dissolve one tablet of Roche cOmplete protease inhibitor cocktail (Sigma Cat# 11697498001) in 2ml H2O. Aliquot and store at -20C.
0.1 M NaF Phosphatase Inhibitor (100X):
Dissolve 0.21 g NaF (MW 41.99) in 50 ml H2O. Aliquote and store at -20C.
0.5 M Na3VO4 Phosphatase Inhibitor (250X):
Dissolve 4.6 g Na3VO4 (MW 183.9) in 50 ml H2O. Aliquote and store at -20C.
10% Sodium-deoxycholate:
Add 1 g Sodium-deoxycholate in 10 ml H2O. Wrap with alumna foil and put on rotator to dissolve. Store at RT, protect from light.
RIPA Buffer:
50 mM Tris-HCl, pH 7.4
150 mM NaCl
1% NP-40
0.5% Sodium-deoxycholate
5 mM EDTA
0.1% SDS
RIPA buffer
1 ml
1 M Tris-HCl, pH 7.4
50 ul
5 M NaCl
30 ul
10% Sodium-deoxycholate
25 ul
10% NP-40
100 ul
0.25 M EDTA
40 ul
10% SDS
10 ul
H2O
665 ul
0.1 M PMSF
10 ul
0.5M Na3VO4
20 ul
0.1 M NaF
10 ul
25X Cocktail
40 ul
2X SDS Sample Buffer
4% SDS
50 mM Tris-HCl, pH 6.8
50 mM DTT
10% glycerol
1% beta-mercaptoethanol
0.02% Bromophenol blue
Note: Sample buffer can to be stored at -20C for up to 1 month to preserve DTT.
Blocking Buffer
5% non-fat dry milk in PBS-T. Good for 1 days at 4C.
