The dynabead protocol suggests to use at least 100ul of antigen-containing lysate to resuspend the bead in immunoprecipitation. I usually 10ug of lysate dilluted in PBS to get 100ul and give me good result, but I also need to use supernatant (no-ip protein) for Western blot analysis, and 100ul is too diluted.
Have you ever had experience with using dynabead/maneticbead in IP with lower volume of antigen-containing lysate?
Thank you very much
They suggest 100ul because thats the minimum you required to have efficient mixing of beads in solution upon rotation. I have tried lesser than that, it works but does introduce some variability. However, the solution i banked upon to test the Supernatant, is to TCA precipitate the protein from 100ul and resuspend in minimum volume for analysis. That has always worked reproducibly for me and never given me problems.
They suggest 100ul because thats the minimum you required to have efficient mixing of beads in solution upon rotation. I have tried lesser than that, it works but does introduce some variability. However, the solution i banked upon to test the Supernatant, is to TCA precipitate the protein from 100ul and resuspend in minimum volume for analysis. That has always worked reproducibly for me and never given me problems.
antigen volume less than 100ul would definitely hamper the antibody binding in your rotating wheel. I'm just wondering why antigen volume is your constrain? you can always make up the volume with IP buffer. As you'll be pulling the complex, larger volume should not matter at all. All the best
Use un-diluted lysate as input and diluted lysate for IPs. Unless I am missing something here, I don't see any problem with it.
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