Hi everyone,
I am doing co-IP between two proteins. When I pulldown protein A, I find protein B in the test sample AND the mock sample.
The first picture is my first blot, then the second is with more washing steps. Could this be caused by non-specific binding of the beads or the protein A antibody?
Here are my reagents.
Beads: Pierce protein A magnetic beads.
Primary antibody: Normalised rabbit IgG
(https://www.merckmillipore.com/GB/en/product/Normal-Rabbit-IgG,MM_NF-12-370)
Hi,
try this:
repeat the experiment.
Additionally, load normalized rabbit IgG (the amount you use for experiments) mixed with loading buffer (no lysate), and the Ab you use for IP of protein A (the amount you use for experiments mixed with loading buffer (no lysate) - these should produce negative results on WB but sometimes they bind 2°Ab at different sizes, not including standard heavy and light chain. Also, try everything without an antibody to see if your beads collect something unspecific from the lysate. This should remove any doubts you have about your results.
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