Freeze/Thaw procedure in cell lysis - How is this done??? (Jul/10/2006 )

Ok,, sounds good, at least I know some one tryed it already, hehehe.
Thanks

QUOTE (medchemgirl @ Jul 11 2006, 09:06 PM)

I am making RIPA from scratch, I won't have manufacturer instructions, but I dont think I will be adding lysozyme and DNase.

QUOTE (kylvalda @ Jul 11 2006, 03:27 AM)

QUOTE (swanny @ Jul 11 2006, 08:52 AM)

You can also add lysozyme and DNase to your lysis buffer, both of which speed the lysis /genomic DNA shearing steps, sos yuo get to move on to the fun bits, liek purifying the protein...

as RIPA buffer contains much detergents (SDS) most of enzymes aren't acitve in it
(the DNAse we use for examples isn't active in RIPA) -
so you have to check manufacturer's description of enzyme activity

I tried adding DNase though manufacturer said it wouldn't work and it made no difference on the gl so just leave it be...

we prepare whole cell extracts using ripa buffer
452.5 ml dH2O to final 500 ml
25 ml 1M Tris HCl pH 8 to final 50 mM
15 ml 5M NaCl to final 150 mM
5 ml NP-40 to final 1%
25 ml 10% deoxycholic acid to final 0.5%
5 ml 10% sds to final 0.1%


before use for each ml we add
10 ul 0.1M NaVO3 to final 1 mM
1 uL 1M DTT to final 1mM
protease inhibitor (1 tablet for 10 ml)
and 10 ul PMSF 100 mM to fianl 1 mM


doesnt require sonication or F/T
just incubate rotating at 4C for 30 min, spin 30 min 10000 rpm , taake supernatant and estimate protein for your western

I get very poor protein yields with F/T in the buffers I tried... (Tris HCl, HEPES) this because I wanted to assay enzyme activity

Thank you, I will try this recipe. I appreciate it very much!!!