I need to perform IP for a target which binds to lipid droplets. I used RIPA to prepare cell lysates for western blot which showed good results. But not sure whether it is good for IP.
The only way to know is to do the experiment. Include a positive control (non-IPed) on your gel. You could also try preparing a sample with a less aggressive buffer and compare (ie, 1% TX-100 or NP-40)
RIPA buffer is quite harsh. For IP, you need fresh lysate made with an actual IP lysis buffer. Myself, I use an IP lysis buffet made with 50 mM HEPES (pH = 7.5), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2, and 1.0 mM EGTA. Good luck!
Thanks. Is your lysis buffer for Co-IP or just IP? At this stage, I just need to pull down one target, not a complex, from lipid droplets.
Hi Wensheng,
there are a few different recipes for the RIPA buffer, more or less stringent. Anyway, whatever you used, we have done IPs in a quite stringent RIPA buffer (1% NonidetP-40, 0.25% Na Deoxycholate and 0.1% SDS) and we could very efficiently IP and co-IP our protein and the known ligands. I guess you have to try with your specific Ab, but of course the higher the SDS concentration, the more unlikely you will succeed.... although, if you are working with a protein in lipid droplets, I guess some SDS you'll have to use to extract it.
Good luck.
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