Dear All,
I performed the Co-IP to see the binding and co-localization of STAT3: and NFkB p50 in Human PBMcs
Co- immunoprecipitation were performed on PBMCs (4×106) from cells treated with and without IL-6. Co-IP experiments results showing that NFkB p50 is co- immunoprecipitated with STAT3.
My queries are:
1. In my co-ip experiment, I pull down with NFkB p50 antibody at 4 degree overnight incubation, then after I used STAT3 (MW 78kD), antibody as a primary antibody in the western blot.
After developed the blot through ECL, I got a bands around 78kD.
My exact question is how we find the co localisation of STAT3 &
NFkB.?? Did I want to one more band of NFkB p50 subunit in Blot?
I attached the blot picture.
Please help me out.
If you probe with STAT3 antibody after Co-IP, and if your STAT3 Ab is specific then you should not see any NFkB band. You also need to perform reverse-IP, mean you need to perform pull down with STAT3 antibody and then probe with NFkB p50 subunit antibody.
Hi,
IP: When you pull down with NfKb then blot with STAT3 ..you should see only STAT3
Similarly,
Reverse IP: when you pull down with STAT3 and immunoblot for NfKb...Only the desired Nfkb subunit should be seen.
You have to perform both : IP and reverse IP, to confirm binding between the two antibodies. You can also perform immunoblotting , just to visualise the colocalisation..(though this is a qualitative proof, the confirmatory being the immunoblots for IP)
All the best !
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