Confirming IP with WB - Can I use the same antibody for both IP and WB? (Oct/12/2007 )

Hi,

I did an IP and then tried to confirm that I've purified the protein using WB. But I have only one rabbit derived antibody. So I used that for both IP and Western.

The IP was done using protein A beads (the Ab was not covalently linked to the beads directly)... consequently the primary Ab also eluted out and showed up as a very intense band in the Western... so much so that I could not even see my protein of interest clearly. Sure, I could reduce the amount of Ab used for IP next time.. but I'm wondering if there's any other way to reduce this problem.

For example, after protein transfer to membrane, can I let the primary and secondary Ab bind to each other in a solution separately first and then allow the complex to bind to the protein on membrane? That way, I can have excess primary Ab compared to secondary Ab.... so there should not be much of free secondary Ab that can bind to the primary Ab which ran on the gel. Any suggestions.... or alternatives??

Thanks!!

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you need two different Ab´s: one for immunoprecipitation and one for Wb; if protein is overexpressed and carries a tag, you may use one Ab against the tag, the other Ab against the original protein...;

if you use the same Ab for IP and Wb, you will definitely see an enrichment

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Try Protein A HRP as your 2nd Ab. That works on me.

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Hmmm... sorry, I guess I didn't understand you. If I use protein A HRP as 2nd Ab, then it will bind to any IgG type molecule and that includes the primary Ab that ran on the gel. So I will still see a band for primary Ab. Can you please clarify? Thanks.

The only possible solution I thought was to use two different Abs as The Bearer said (for example, mouse Ab for IP and rabbit Ab for WB) so that the secondary Ab in Western detection will not cross react with the Ab from IP. But still not sure if anti-rabbit will not entirely cross-react with mouse Ab. I just want to know if any of you have alternative suggestions (or if the possibility I mentioned in the original post will work).

Thanks!!

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Yes, here is a paper addressing this question.
"Clean western blot signals from immunoprecipitated samples"
http://www.pubmedcentral.nih.gov/articlere...i?artid=1350844

And also, the TrueBlot system from eBioscience is good.

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Thanks... that makes sense. I appreciate your response.

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