RIPA recipe with Bradford compatibility..., - (Apr/28/2011 )
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
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,
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