FAQ
Protein expression is a complex regulatory process, and the detection of protein expression may be greatly related to the specificity of the antibody. If infection or transfection efficiency is excluded, for no overexpression effects, the possible reasons include:
① The background expression of the target gene in the cells is high, making it difficult to achieve a significant overexpression (the basal level can be detected by qPCR).
② The target antibody is not specific, the protein has different isomers, making it impossible to detect significant overexpression with the target antibody.
③ The target gene is highly related to cell growth and proliferation, and if overexpressed excessively, it may affect cell status, preventing normal growth and proliferation, and the cell itself may regulate to reduce or even restore the overexpression to normal expression levels.
It is suggested to try detecting with tag antibodies or qPCR methods. With tag antibodies, it could be detected exogenous overexpressed proteins.
For knockdown studies, the possible reasons for not detecting interference effects include:
① The background expression of the target gene in the cells is low, making it difficult to achieve lower expression (the basal level can be detected by qPCR).
② The target gene is highly related to cell growth and proliferation. If knocked down excessively, it may affect cell status, preventing normal growth and proliferation. Furthermore, the cell itself may regulate to reduce or even restore the knockdown effects to normal expression levels.
③ For specific promoters, if cannot be limited to specific tissues or cells, then the knockdown effects may also not be detectable. It is suggested to use in situ hybridization or immunofluorescence.
④ The target antibody is not specific, the protein has different isomers, making it impossible to detect significant knockdown with the target antibody. It is suggested to try detecting with qPCR methods.
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