I am currently trying to precipitate a protein for which I have an antibody targeting a peptide epitope. However, so far it seems likely that the epitope is buried.
I have heard that it is possible to do IP's under denaturing conditions, exposing any buried epitopes. This got me quite excited as it might be just what I need to precipitate my protein.
But I am wondering how do you avoid denaturing the antibodies during such a denatured IP? If the denaturing agent is in the present in the extraction buffer it will denature the antibodies during the binding step. However, I would suspect that removing the denaturing agent would result in refolding or worse.
Hi ViK,
I agree with Arjan. You have first to confirm that the antibody do recognize your protein in western blot. You can also try his suggestions.
If you want to try denature your protein, then You can make your protein extract in RIPA buffer with 1% or even 2% SDS and then dilute it 10 times with RIPA buffer without SDS to bring your sample to 0.1 or 0.2 % SDS final concentration, and then perform IP. Antibodies can withstand 0.1 and 0.2% SDS.
it is not easy to make refold a denatured protein but there are exceptions. Also, as one of my former bosses once told me “You come up with a 99 reasons for why you shouldn’t do the experiment and not one why you should do it. Just do it”
Good Luck
Thanks guys! Really usefull advise.
I have used the antibody for western blots and got nice bands, so I know it binds to the protein.
I think I will go with the SDS dilution to see if I can pick it up, and then ultimately combine it with in vivo crosslinking to isolate any interaction partners.
You could denature your sample (e.g. in SDS or urea buffer) and then dilute it for the IP such that the antibody is not affected. We sometimes use a protocol for FLAG-IPs where the cells are scraped from the dishes, lysed in a buffer with 1.85 M NaOH + 7.4 % β-Mercaptoethanol, then precipitated with 50 %TCA and then resuspendend in 0.5 M Tris, 6.5 % SDS, 12 % glycerol, 100 mM DTT. From this mess we take 300 ul and dilute it ti 14 ml in RIPA and do the FLAG-IP with 50 ul anti-FLAG matrix.
It sounds harsh, we loose a lot but we preserve many post-translational modifications. Maybe it also helps in your case.
But I'd also try WB first. Some people here use PVDF and boil (!) the membrane after blotting to fully denature the bound proteins.
Yes, the above comments are very sensible. First check the antibody in WB, then use a harsh lysis buffer and dilute or dialyze before adding the antibody. Polyclonal antibodies (also peptide-generated and affinity purified) should work fine at 0.1% SDS. So if you start at 1% SDS in RIPA buffer and dilute the SDS down (not the other RIPA components!) to 0.1%, you should be fine. You may even have to heat your lysate at 1% SDS in the presence of a reducing agent as discussed above. Make sure to dilute the reducing agent as well when using this approach.
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