My hypothesis is Protein A and Protein B has interaction in cell. So I prefer to co-IP.
About the strategies, which one should I choose?
1 Use beads covered with Anti-Protein A, pull the AB off, then use the anti-Protein B for testing?
2 Transect the cell with Flag-Protein A, use beads covered with anti-Flag, pull the Flag-AB off, then use the anti-Protein B for testing?
If I choose strategy 1, should I use IgG instead of anti-A as negative control?
If I choose strategy 2, which negative control should I use?
1 Cell transfected with blank Flag plasmids?
2 Cell transfected with un-flag normal Protein A?
3 Cell transfected with Flag-IgG?
Both strategies would work. I would choose between them based on how good you think your antibody against protein A is. If the antibody is really great and works well in IP, I would chose strategy 1. Strategy 1 is the most natural and does not require modifying protein A with a tag. Tags can sometimes modulate the behavior of the protein they are attached to, if you are unlucky. Adding tags can, for instance, affect the protein's trafficking or function.
(possible downside is that if your antibody happens to bind protein A in the same region where protein B is supposed to bind, you might destabilize or disrupt that interaction. In that case you would only pull down protein A and probably misinterpret the result).
If your antibody against protein A is rather low affinity and does not generally work really well in IP, I would rather go with strategy 2.
The negative controls that you propose are all good controls.
1) Both strategies would work and you should try both. But more importantly, you should do the reciprocal experiment, pull down protein B first, and see if you can co-precipitate protein A. This is what a reviewer will ask. Reciprocal IP is the gold standard in protein-protein interactions.
2) It also depends how you detect after IP. When you use in vivo labelled cells to extract proteins (including protein A, protein B and AB complex), then the detection after IP is using autoradiography. You use the known molecular weights of protein A and protein B as indicators of the right bands. If you can dissociate the complex by changing the conditions (i.e. add ATP, or add Calcium, of change the pH), you can do IP, then release, and re-immunoprecipitate from the release-buffer supernatant with the other antibody.
If you detect with western blots, then you must be aware of the fact that your boiled sample contains your antigen and antigen complexes, but also the primary antibody and protein A. Your negative controls should be designed to interpret your western blot and rule out detectionn of the primary antibody bands and protein A of course.
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