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Protein Lysates from Cells in Culture

Cell Lysis Buffer

5mL 0.1M Tris HCl pH 8 (10mM)
0.44g NaCl (150mM)
0.02g EDTA (1mM)
0.5mL nonidet P40 (1% w/v)
0.05g SDS (0.1% w/v)
Make up to 50mL with MQH2O and filter sterilise.
Store at 4 degrees C.
This cell lysis buffer has a limited shelf life (at 4 degrees C). I recommend making fresh buffer up annually. My own personal experience has been that after ~18months my protein lysates began appearing very distorted following SDS PAGE and were no longer usable (whether fresh lysates were made from >18month old buffer or lysates that had been stored at -80 for >18months were used).

20x Complete mini Protease Inhibitor

Take 1 tablet and dissolve in 0.5mL MQH2O by pipetting up and down.
Store on ice.

Dilute 20 fold in cell lysis buffer

Cell Lysis Buffer containing 1x Complete Protease Inhibitor

50uL 20x Complete protease inhibitor
950uL Cell Lysis Buffer

Confluent 6 well (9.6cm2) lyse in 150uL cell lysis buffer.
Confluent T75 lyse in 800-1000ul cell lysis buffer.
If you choose this way you will need to quantitate your protein lysates to enable loading equal total cellular protein amounts onto your gel. I have followed this method and have found it to work well.