5 replies to this topic

[guyan]

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.

[madrius1]

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.

[guyan]

madrius1, on 13 November 2010 - 10:56 AM, 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.

[Curtis]

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

Edited by Curtis, 13 November 2010 - 11:20 PM.

[guyan]

Curtis, on 13 November 2010 - 11:19 PM, 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.

[Sweetsugar70]

Thanks try it here too,
Godspeed!


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