Hi I am working on immunoprecipitating AMPK alpha protein from my lysates. I am using a sigma aldrich IP kit that includes beads mixed A+G proteins.
I tried 3 different elution techniques: Glycine, citric acid and SDS. All 3 worked and gave me a specific band on western blot (of course SDS was stronger).
The issue is that with the 3 elution methods I am getting antibody contamination so I see the heavy and light chain. I used the beads only as negative control theere was not antibody contamination on my blot so I knew it is the antibody getting eluted with my antigen.
Could you please advise on tricks you may have to avoid this contamination?
The antibody is not covalently bound to the beads, so it will elute under harsh conditions such as low pH or SDS. You can avoid detecting the antibody chains on the blot by probing with an antibody from a different species than the one you used for the immunoprecipitation, so that the secondary antibody will not recognize the IP antibody.
Covalently coupling the antibody to beads is another way to keep from eluting it. You could use CNBr-activated Sepharose, for example.
You may want to try Amino-Link beads from Thermo FIsher. When we used these beads a long time ago, we could isolate protein of our interest free of IgG. Image is attached for your information.
if all those suggestion don't work still, try to run a gel with a different concentration which you can separate your protein from the antibody contaminant, depending on the molecular weight of your protein and the antibody you could run the gel a bit longer or have a gradient concentration gel (SDS-PAGE) that allow a better separation between your protein and the antibody. All the best.
Another method I use is cross-linking the IgG antibody to the protein A resin using DMP before Immunoprecipitation.
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