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[GNANA]

Hi everybody...,
I need a recipe for RIPA cell lysis buffer...,the one which i used is not compatible to quantify using bradford assay(SORRY I DNT HAVE OTHER OPTION TO QUANTIFY CURRENTLY),

The recipe which i used (for 100ml)...,

5ml 1M tris Hcl (ph 7.4)
3ml 5M nacl
1ml 10% NP40
0.5g sodium deoxycholate
1ml 0.5M EDTA
0.2ml 0.5M EGTA
0.1% SDS

i got the recipe from this forum, this is good but couldnt quantify using bradford assay...

Please suggest me a recipe (but stronger like RIPA) with bradfdord compatibility.....

Thanks in advance,
Gnana....,

Edited by GNANA, 28 April 2011 - 09:20 AM.

[proteaMatt]

Your biggest challenge in finding a lysis buffer that will work as is to both lyse cells (with the efficiency of RIPA) and be Bradford compatible is the concentration of surfactants. I have recently been testing our ProteaPrep Coomassie Protein Quantitation Kit (our version of Bradford assay) with various surfactants including NP-40 and SDS. From my experiments 0.1% NP-40 doesn't impact the results too much, but even 0.1% SDS really lowers the reliability of the data. I see that your RIPA recipe includes both 10% NP-40 as well as 0.1% SDS, to allow this buffer to work with a Bradford assay I would expect you would need to dilute your sample at least 10x.

I don't have too much experience with RIPA buffers so I can't really recommend an alternative recipe. Something you could try is a lysis buffer like ProteaPrep Cell Lysis Kit, Mass Spec Grade which contains an acid labile surfactant that can be degraded prior to the Bradford assay so that your results won't be skewed. The only caveat that I would make is that this lysis kit is not going to be as efficient at lysing cells as a RIPA buffer, but from my experience it does a good job.

The three possible solutions that I see to your problem are:
1. dilute your lysate so that surfactants won't interfere (try this first, it will probably work)
2. try a labile surfactant instead of NP-40 and SDS
3. try the Lowry assay

Edited by proteaMatt, 12 May 2011 - 06:21 AM.