My cell line has endogenous expression of Mol.A and B. But, when I overexpress A, I want to know whether it is altering endogenous expression of B or not. What experiment should I conduct to resolve this?
Hello, you could maybe try to compare/quantify the amount expressed using a Western Blot. Will not give you very accurate numbers but might be worth a quick go.
Beside Western blots and q-PCR, you can try confocal microscopy imaging for proteins' localization, co-localization, appearance and intensity signals. It can be done both on a single-or multiple-cell levels. They also give nice pictures for publications :)
Firstly, you should investigate wherether there are interaction beteween A and B through BIFC or yeast two-hybrid system. Secondly, you identify if A bind the promoter of B. or, A do not directly bind the promoter, but through other genes. last, you can test the expression of the A and B through qPCR.
You could also use an siRNA for A and analyze if B diminishes.
Hello, first things first! you need to have your controls. 1. if you want to determine whether the molecule A, directly or indirectly affects the expression of the molecule B, than you need to have a control of the same cell line, cultured in the exact same way and transfected with a negative control that is not supposed to alter the expression of molecule B. Only then can you tell that mol A affects mol B (by quantifying the expression of you protein B with qPCR, western blot or luciferase). 2. Now if you have established that mol A affects the expression of mol B, and you're looking for a direct interaction between both proteins, you can do colocalization experiments or yeast two-hybrids (both a way more time consuming than qPCR or western). Hope it helps!
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