Which lysis buffer should I use? - (Nov/13/2010 )
My protein of interest is a chromatin associated one. I tried IP150 (NaCl 150, NP40 1%), IP300 (NaCl 300, NP40 0.2%) and RIPA buffer to extract it (I sonicated when I used the first two). Only RIPA buffer can efficiently extract the protein. But the RIPA buffer is not so good a choice for my followed IP. What should I do then? Looking forward to some replies.
You could look at ChIP protocols, skipping the crosslinking part. It is essentially a transcription factor or DNA bound protein IP and it works like a charm.
madrius1 on Sat Nov 13 18:56:00 2010 said:
You could look at ChIP protocols, skipping the crosslinking part. It is essentially a transcription factor or DNA bound protein IP and it works like a charm.
I would like to use anti-Flag M2 resin for the IP, which is not so stable in buffer with SDS.
have you tried RIPA yourself to say this? or you just read in manuals? However I also don't like RIPA because it's very harsh.
you can try CHAPS 2%, with HEPES and NaCl. trust me it's way better buffer than RIPA.
I'm working with the same anti-flag m2 resin, it works fine with CHAPS
Curtis on Sun Nov 14 07:19:25 2010 said:
have you tried RIPA yourself to say this? or you just read in manuals? However I also don't like RIPA because it's very harsh.
you can try CHAPS 2%, with HEPES and NaCl. trust me it's way better buffer than RIPA.
I'm working with the same anti-flag m2 resin, it works fine with CHAPS
thanks, I will try it.
Thanks try it here too,
Godspeed!