I am doing my research on protein interaction in different cancer cells in response to different drugs.
I am performing IP to check whether my protein 'X' is interacting with p38gamma, treatment with Adriamycin and etoposide. Pulling down with my protein 'X' primary antibody, I could see the interaction of p38gamma with my protein 'X' in both Adriamycin and etoposide treatment. But, when I reversed the reaction i.e., pulling down with P38 gamma antibody, I could see the interaction only with the etoposide not with Adriamycin treatment. (I repeated twice and confirmed the same results.)
Is this situation possible? Am I doing some mistakes?
Perhaps under conditions of etoposide treatment the interaction of your protein X with p38gamma is slightly stronger (i.e. more stable complexes form) or of different nature. If you have an awesome high-affinity antibody directed against protein X, this antibody might pull down p38gamma under both conditions. If the p38gamma antibody, however, is of lower affinity or otherwise suboptimal for IP, you might lose weak interactions.
We were discussing the same point, i.e., perhaphs there is a threshold level of interaction, either P38 gamma antibody is not pulling down efficiently or it could be the amount of protein we used might be less. But, I already used 1mg protein which is enough though. we wonder is there any contextual binding of these protein in different stress situation, perhaps we are completely wrong..
Thanks a lot for the suggestion Dr. Ralf Leonhardt , highly appreciated !!!!!
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