Hi all,
I have some trouble in over expressing membrane receptor protein in HEK cell.
I am studying membrane receptor protein. So I want to knock down this protein via shRNA and to see what will happen in vivo. So I am planing to test various shRNAs in target protein over expressed HEK cell. However the problem is the target protein expression. I cloned the target gene to plasmids for mammal cells expression, but I cannot get this protein band on Westernblot. I taged with c-Myc at N terminal, I also couldn't get western blot band. I think transcription is fine because after the target gene there is IRES and DsRed and I can see red fluorescence after transfection.
Is there any specific strategy for membrane protein cloning?
Can you provide more detail about what you are doing. Best wishes
I agree with Richard. We had a similar experience with the Myc tag being at the N-terminal end of our target protein and were unable to detect the protein expression by Western blotting following transfection into 293T cells. We then put a FLAG tag at the C terminal end and detected the protein using an anti FLAG antibody. So far, we still do not know why the protein remains undetectable when an anti Myc antibody is used in Western blotting. Have you tried using an antibody against your target protein?
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