Total protein extraction

From Genome@Yale

Following protocols from Pierce are available:

• B-PER - bacterial extract
• M-PER - mammalian cell extract
• NE-PER - nuclear extract
• BCA - protein concentration

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Total lysate using RIPA

Prepare RIPA and store at 4C

                     for 50mL		for 500mL
50mM Tris, pH 7.4    2.5mL		25mL 1M	
150mM NaCl           1.5mL		15mL 5M
0.5% Deoxycholate    0.25g		2.5g
0.1% SDS             0.5mL		5mL 10%
1% NP40              5mL		50mL 10%
dH2O                 45.5mL		455mL

Add protease inhibitors (1 Complete tablet per 50mL RIPA)

For 5 million cells, add 1mL of RIPA buffer to lyse and transfer lysate to tube

Vortex 30 seconds.

Centrifuge to pellet the insoluble and transfer to new tube

Store extract at -80C.