Hi,

If this what you meamt ' Can you do IP with the antibody specific for your protein'

Then definitely Yes. You need to couple your antibody with protein A/G sepharose beads and do IP. You will get you target protein and the other proteins that are interacting with it, as long as the  protein-protein interactions are not completely abolished by the detergents.

I hope that helps

Hi Ashish,

I understand your question is whether you can immunoprecipitate a protein using an antibody that works for Western-blot. My answer is "depends": For IP, the antibody has to be able to recognize the epitope when the protein of interest is in native conformation. In SDS-PAGE Western-blots, proteins are denatured and the "denatured" epitope that the antibody is detecting in WB may or may not be accessible in the native conformation of the protein. 

I suggest trying the antibody for IP using between 1-3 ug and the proper protein A/G depending on the type of IgG of the antibody and using a relative mild buffer (detergents such as TritonX100 or NP40 are mild and break protein-lipid, lipid-lipid associations but not protein-protein interactions and do not denature the proteins).

I hope this helps,

Ana

I agree with Ana, but would like to add that if your antibody works in other applications that require the detection of native protein (e.g. flow cytometry, immunofluorescence, cryo-immuno EM, native gels etc.), then it is at least pretty likely that it will work in immunoprecipitation as well. Immunoprecipitation, however, often requires a little higher affinity of your antibody for its target protein, because the interaction has to survive several washes (I usually do up to five).

Oh, and make sure you choose the correct beads. Not all isotypes from all species bind equally well to protein A or protein G. Make sure you use the best beads for your specific antibody.

Hello, Ashish,

First of all, usually there is an information about applications of antibodies on a manufactory's website. So, if there is an "IP, ChIP" in the list of applications, it's likely that Your antibodies will work in ChIP.

And of course, You can check this antibodies in small IP reaction (in small volume like 10ul). Take supernatant after incubation with sepharose, and elution, and check it with same antibodies on western blot (you must divide antibodies and protein from each other in SDS-PAGE, becouse You'll have a signal from Your IP antibodies). Controls for western blot - Your probe before IP and separately antibodies, and You'll can say - where is Your protein and Your antibodies in IP reaction.

Good luck!

I guess Ana and Ralf already gave you most of the information. Just keep in mind that Abs that work in IP recognize an epitope that is exposed on the surface of the natively folded protein. These surfaces are also the ones used in protein-protein interactions, and since the Ab-epitope interaction is very strong, if it happens to overlap with the binding domain to a ligand it will displace the ligand, so you will not be able to detect it in the WB. Take home message: if you will be able to IP your protein and detect its ligands (what you call "downstream proteins"), fine. If you will be able to IP your protein but will not detect one or both ligands there is not much you can say, you will have to address the problem in a different way (for example IP the ligand and check for your protein, or do a pull-down, if you can produce your protein in vitro and bind it to beads, or tag your protein with GST/Strep-tag/etc, transfect it and pull it down taking advantage of the tag).

By the way, sometimes Companies reveal how the Ab was produced and if the structure of your protein is known you might be able to know what to expect...