I have been using Dynabead protein-G for immunoprecipitation of my target protein in a mix of antigen from two different protein samples. My target protein X is supposed to bind to the protein Y in the in vitro assay. Protein Y is tagged with 6x His and hence I am using anti-6x His to immunoprecipitate the target i.e. X. The input gives a nice band in western blot but immunoprecipiated bead sample shows a downward shift from 5kDa. I am also getting the heavy fragment band in both pre cleared sample and IP sample. What could be the reason?
Hi,
Usually the input has large protein complex compared to the IP eluate, So the input runs slowly compared to the IP samples in SDS PAGE. I had similar issues but as longs ur antibody is specific you dont need to worry about the 5 KDa difference. Another thing if ur membrane is not straight that could also be a reason. Better do ponceau staining before blocking, so you will be sure everything is fine.
Your next question about the heavy band, when using beads coupled with antibodies for IP, you usually get heavy and light chain fragments from your IP antibodies. To avoid that you need to covalently couple the antibodies and you have to elute without breaking the coupled antibodies, the most simple way is to use antibodies with single chain (antibodies from Ilama, you can check with chromoTek for anitbodies that are coupled to the beads). These antibodies will not give the heavy fragments.
Good luck with your work
Hi Hakim,
Great!!
I completely agree with your explanation of heavy protein complexes moving slower. Another step that I am going to take is that I will cut the bands out and probe for the presence of target protein using MS. Alternatively, I will also strip the membrane and reprove them using antibody against 6x His. This will confirm the existence of X and Y in the same protein complex.
Hi Alok,
If you can show by MS that more than sufficient that you have them in a complex. But include proper negatively control to rule out the fact that your X and Y are binding to the beads. You can also do a direct binding pull down study which could answer X and Y are interacting directly or not. If Yes it is a nice result , if NO then you know they are atleast in a complex.
But I also would like to point out that the IP and precleared lysates are moving faster than input that's why they are shifting downwards. Please see the question.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Immunology Techniques