Cell Lysate Preparation Protocol

FOR MONOLAYER CELLS

1. Rinse monolayer cells 3-4 times with PBS.

2. On the final rinse, aspirate as much PBS as possible and add 5 ml of ice-cold PBS containing 0.5 mM EDTA and use a cell lifter or cell scraper to bring cells into suspension.

3. Transfer suspension into a 50 ml centrifuge tube and add 5 ml ice-cold PBS to flask.

4. Centrifuge cells at 1,500 rpm for 10 minutes at 4°C and aspirate supernatant.

FOR SUSPENSION CELLS

1. Centrifuge suspension at 1,500 rpm for 10 minutes at 4°C and aspirate supernatant.

2. Re-suspend pellet in 15 ml PBS and centrifuge at 1,500 rpm for 10 minutes at 4°C and aspirate supernatant. Repeat 2 more times.

3. For every 1 x 106 cells add approximately 100μL of ice-cold Lysis Buffer with 1X PIC (see recipe below) and re-suspend pellet, ensuring no clumps remain.Incubate on ice for 60 minutes.

4. During incubation time, transfer contents of each tube to a microcentrifuge tube.

5. Centrifuge at 13,000 rpm for 30 minutes at 4°C.

6. Collect supernatant into an appropriately labeled tube and determine protein concentration using the BioRad modification of the Bradford assay.

7. Re-suspend the cell lysate is in 2X SDS sample buffer (see recipe below) by combining equal volumes of 2X SDS sample buffer and cell lysate. Heat at 95-100 °C for 3-5min.

8. Use immediately or aliquot and store at -20°C or -80°C. If storing lysates, warm prior to loading on SDS-PAGE

BUFFERS

1X Lysis buffer: 10 mM Tris, pH8.0, 130 mM NaCl, 1% Triton X-100, 10 mM NaF, 10 mM NaPi, 10 mM NaPPi.

50X Protease Inhibitor Cocktail (PIC): 800 mg/ml benzamidine hydrochloride, 500 mg/ml phenanthroline, 500 mg/ml aprotinin, 500 mg/ml leupeptin, 500 mg/ml pepstatin A, 50 mM PMSF. Make 50x solution store at -20°C.

2X SDS sample buffer: 120 mM Tris-Hcl (pH 6.8), 20 mM EDTA, 4% SDS, 0.06% Bromophenol Blue, 20% glycerol, 0.4% beta-mercaptoethanol