I have been working on two proteins. And I think that these two proteins interact with each other. To prove that, I analyzed there association by endogenous IP, but unfortunately I could not get any band in the western blot after pull down.
Here my questions is:
1) Is there any possibilities that endogenous IP could fail to find association ( And expressed IP could get this association).
This depends on many factors like the expression level of the 2 proteins , nature&stability of interaction , conditions of CO-IP , Specificty and senstivity of the antibodies,.........
Sometimes there is an endogenous interaction but it is hard to be seen without overexpressing your target proteins or at least one of them.
I am intrigued to know the levels of expression of these two proteins in the input samples. How strong are the signals? If the signals in the input sample are very low, then that could be a point of concern for detection of their endogenous interaction by Co-IP. Then the question of other troubleshooting strategies paves in.
It could happen when the epitope of your protein is closed when being in complex. In any case you should see the band, you can make a control western from the crude extract to see whether you have a protein.
Dear Nupur Raychaudhuri,
I use chemiluminescence for the detection of blots. For protein A, I could easily get the band at a low exposure time, while it takes longer exposure time to detect protein B. From this results, I do not know whether I am right to say that the expression of protein B is low.
I just suspect that the protein B concentration is quite low to be detectable. I see most of the papers use expression vectors for this type of experiment. So, I have ordered vectors for these proteins, and will try with those expression vectors.
Thank you all for your comments and suggestions.
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