I want to determine whether protein A and protein B interacts with each other by co-immunoprecipitation analysis. For that I prepared Myc-tagged protein A and Flag-tagged protein B plasmid DNAs. I transfected these plasmids in HEK293T cells, and after 48 h, I lysed the cells and preformed co-immunoprecipitation.
I found no interaction between these two proteins.
As HEK293T is a human cell, and my proteins are of mice origin, my question is that if these two proteins have a indirect interaction, is it possible to detect this interaction by IP.
Additionally, if the interaction needs some signal (eg. stimulation), is it appropriate to use HEK293T cells, or do I need to use mouse cells.
In fact, the best way would be another:
a) use another more adequate murine cell line or (better)
b) use another more adequate testing methode (e.g. a yeast-two-hybrid system etc.)
I overexpressed Mouse proteins in 293T cells and performed IP experiments without any problem, but those two proteins are interact directly.
Dear Dr Palikhe,
I like yeast hybrid ideal too.
Could you make proteins with human cdna's? And retest with all human cell and proteins.
Also you are most likely using bovine, cow serum for cell tissue cultures. This isbanother non human routine dogma.
You did find the human cells and mouse a&b did not have a reaction. This question was bound to be asked, and you answered it. Well done sir.
Indirect protein interactions can lead to large screening experimental designs that yield more questions to therom. I have seen this once hunting for an unknown lipid transport protein.
Mouse cells, and mouse serum with protein a and b
Dear all,
Thank you for your comments and suggestions.
I need some time to decide my next step.
I will update about the progress of my experiments.
Regards,
Sailesh
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