Difference between Western Blotting and Immunoprecipitation?

[tonyz06]

Hey,
this may be a stupid question, but what are the advantages of immunoprecipitation then immunoblotting compared to western blotting only?

Does it help get less abundant proteins with IP then IB?

Isn't is not enough to just use western blot for protein levels and regulation of protein level?

[danfive]

Immunoprecipitation followed by immunoblotting will help verify the IP, and identify the band (if more than one) that contains the protein of interest, the IP will also deplete or wash away the major non-specific proteins which is very useful if you are going to use MS for further protein ID.

If purity and protein isolation are not necessary, then most likely Western Blot will work best. The only exception I can think of is if the protein is very large or hydrophobic or generally has trouble migrating on the gel---with this situation monitoring a single (Immunopurified) band on the gel will give you an easy test for protein migration on the gel.

[MiraReynaWhite]

Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. So you must be able to produce at least a small portion of the protein from a cloned DNA fragment. You will use this antibody as a probe to detect the protein of interest.

Western blotting tells you how much protein has accumulated in cells. If you are interested in the rate of synthesis of a protein, Radio-Immune Precipitation (RIP) may be the best assay for you. Also, if a protein is degraded quickly, Western blotting won't detect it well; you'll need to use (RIP). See the section on RIP for more information, as well as a helpful comparative chart that illustrates the differences between these two techniques.

Immunoprecipitation is a technique that uses antibodies specific to a protein to remove those proteins from solution. The antibody-protein complexes are precipitated out of solution with the addition of an insoluble form of antibody binding proteins. Examples include Protein A, Protein G, Zysorbin, Immunoprecipitin, or the addition of a second antibody to the

[Bonnie]

In theory, IP should be able to detect less abundant proteins compared to WB. However, in my experience, if I can't see a protein very well by WB, I probably won't be very successful IPing it either.

[danfive]

Originally Posted by Bonnie

In theory, IP should be able to detect less abundant proteins compared to WB. However, in my experience, if I can't see a protein very well by WB, I probably won't be very successful IPing it either.

I've done the opposite, On WB I'm limited to gel/well and can only load so much heterogenous proteins, say 80ug/lane. A rare protein would be represented in that mix as a tiny amount.

Now I could use IP to scale up the analysis of the rare protein, I can load much greater amount of hetergenous mix of proteins, lysate, biofluid, cell culture media. and pull down much greater amounts of rare protein. It's all a question of being able to scale up the experiment.

[MoriahToniOwens]

Western blot analysis can detect one protein in a mixture of any number of proteins while giving you information about the size of the protein. It does not matter whether the protein has been synthesized in vivo or in vitro. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. So you must be able to produce at least a small portion of the protein from a cloned DNA fragment. You will use this antibody as a probe to detect the protein of interest.

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.

Western blotting tells you how much protein has accumulated in cells. If you are interested in the rate of synthesis of a protein, Radio-Immune Precipitation (RIP) may be the best assay for you. Also, if a protein is degraded quickly, Western blotting won't detect it well; you'll need to use (RIP)

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