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.
- 2.1. 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.
- 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.
- 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|
|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.
- 5% non-fat dry milk in PBS-T. Good for 1 days at 4C.
- PBS with 0.1% Tween 20