Protein Lysate Preparation Protocol (Culture)

Wash cells using cold PBS followed by ice-cold 1x TNE lysis buffer containing 1% SDS, 0.5% NP-40, 0.5% DOC and protease/phosphatase inhibitors.

TNE lysis buffer:

A	B
Tris	50 mM
NaCl	150 mM
EDTA pH at 7.4	5 mM

Use cell scraper to remove all detached cells and keep On ice for 0h 5m 0s.

Transfer lysates to 1.7mL collection tubes and sonicate for 2 pulses (at 20% amplitude, 0h 0m 10seach), heat for 0h 10m 0s on hot plate (100°C) and centrifuge at 16000x g,4°C.

Transfer resulting supernatants to new tubes (final total lysate).

Perform BCA protein assay following manufacturers instruction and prepare 1xLaemlli samples of standardized concentration for SDS-PAGE.