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Immunoprecipitation vs conventional WB - what are the differences? (Feb/01/2009 )

Hi, folks.

Sorry in advance for my naive question, it's because I've been reading some articles lately with a lot of IP. So here it is: what are the differences between IP and standard WB, regarding results? What are the differences between you precipitating the desired protein with protein G or A and then, detecting it on a SDS gel, and detecting it just with the 1st AB and 2nd AB, as it is in a conventional WB? I know that Co-IP enables one to see protein-protein interaction, but IP just one at a time.
It is, what kind of biological questions does IP answer that WB fails to do?

Thanks in advance for the help.


you can use IP if you want to see something on a particular protein, using an antibody not directed against your protein, but on the post-translational modifications; e.g. phosphorylation.
First we had antibodies directed against the phosphorylated tyrosine of any protein (now there are plenty of antibodies directed against the phosphorylated form of the protein of interest). so we were immunoprecipitating the protein of interest, and then blotting with the anti-phospho antibody.

an other reason to perform IP is when the antigen is weakly expressed. By IP you can concentrate it , and load on a gel.

I don't remember other applications yet.

-little mouse-

Thanks, little mouse.

As you said, today there are antibodies specific for post-translational modifications of certain proteins. For instance, we've already used antibodies for phosphorylated ATM, or histone protein H2AX, without using IP. So, is IP still useful for that? Isn't it more expensive and time consuming?
But for small amounts of proteins, I haven't thought about that condition before.
Thanks a lot for replying.


If you have the right antibody, you don't need to IP.
You would just introduce more troubles.

make a blot with the anti-phospho one, strip, confirm you loaded the same amount in each well by blotting with a normal anti-ATM.

-little mouse-

I see.
Thanks a lot for your tip, little mouse.


you're welcome

-little mouse-

With WB and IP it is important to consider the antibody binding site (epitope) interaction with the antibody. With WB the sample is usually prepared by heating in SDS, this denatures the protein. Some epitopes are conformationally dependent and are destroyed by this treatment. Thus, antibodies that "see" epitopes that are present on denatured protein can be used on westerns (includes majority of anti-peptide antibodies). Antibodies that see secondary or tertiary protein structures usually react only with native (non-denatured) proteins, with these IP is a better choice.
To complicate the matter, some epitopes are "buried" in the protein and are hidden in native proteins. With these westerns are preferable. Often the supplier of the antibody will provide info on the usability of an antibody for IP or western.

Page gels can only be loaded with small amounts of protein (max 5- 10 ug/lane). To detect rare proteins it can be useful to concentrate your sample by IP with one antibody, SDS treat the ppt and load the solubilized precipitated protein on a gel and the "stain" the western with a different antibody.

Hope this helps.


Hey, klinmed,

Thanks a lot. Those are very important information; for sure it helps. Something to bear in mind when working with proteins.
Thanks again you both.