Hi,
I am testing antibodies by immunohistochemistry (IHC) to subsequently use them for immunoprecipitation (IP) of the proteins in question.
Now I did come across two problems:
1. My antibody potentially binds two different isoforms of the protein. One of these isoforms has a RPKM expression of 0.33 RPKM . Do you think expression is to low to be detected by IHC?
2. Is there a expression threshold under which IP does not make sense ?
Thanks,
Eva
A tough question: one can do IP if you are sure that your antibody (polyclonal) recognizes antigen in WB or IHC. IP worked best when we optimized antgen:antibody ratio and time of incubation.
Hope this helps
Thanks for the answer :)
I am pretty sure that for this particular sample I can catch the Isoform which is really abundant but the second Istform with is lowly expressed might be outcompeted.
How exactly do I optimize antigen:antibody ratio and time of incubation ?
How exactly do I optimize antigen:antibody ratio and time of incubation ?
-try to increase the first and/or secondary antibody you use
-some time (depends on the expression level and/or antigen concentration in WB or IHC) the incubation time run overoght even.
Hello Eva!
Are you aiming to get ONLY this second isoform on IP ?
I would first optimize to get the main isoform.
You can keep antibody fixed, and try different dilutions of the antigen, the buffer it is in. Temperature of incubationg with antibody and duration...with or without shaking. Sometimes 2-4 hours at 4 deg C is better than overnight incubation!
My experience:
Very concentrated antigen sometimes gives poor IP results.
You need to wash IP pellet (after Protein A or G to bring down antigen-antibody complex). RIPA buffer followed by milder buffers helps if you have too many non-specific bands.
I heard glass tubes are better than plastic (many proteins adsorb to it). But-I have not compard glass versus plastic to really test this.
Good Luck!
Hi,
thanks a lot for the kind answers!
I am very happy if I get the main isoform!
Okay I will start playing around then.
@Venil The non specific bands occur in western blot right? My current protocol is for IP with subsequent RNA extraction and has only a lysis buffer washing step. Do you think lysis buffer washing is also fine?
btw this is my lysis buffer: 100mM NaCl, 10 mM Tris ph8, 1mM EDTA + 0.1% TritonX (including RNase inhibitors, 1mM DTT + protease inhibitors before use)
Cheers,
Eva
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