Hello all,
I am trying to overexpress a oocyte-specific transcription factor in HEK293 cells. But it never worked in protein level, I cannot detect it through western blot but I do detect it through RT-PCR. So the construct in under control of CMV or EF1a promoter neither of them worked in protein level. When I tried to overexpressed, I have another gene which was cloned into the same vector, it can be detected in protein level by western blot, which can be considered as a positive control.
Then I tried to use lentivirus to infect different cell types, including primary bovine cells, HLP-1 cells and HEK293 cells, still doesn't work the lentivirus promoter is also CMV promoter. I am wondering if anybody have any idea.
Check the agarose gel for the DNA band after RT-PCR. If there is no band of DNA, then your gene is not expressed.
If you will find the DNA band after PCR, but no protein by Western, then it is the antibody.
Mingxiang,
Where did you get your transcription factor insert from? Have you checked the coding sequence? Are you sure that it can be translated when expressed in your vector? Could you add a tag to easily detect any translated product from the start codon?
Philippe Fort Thanks so much for your advice, the insert is from PCR from my cDNA and it fused with FLAG tag and before overexpressed it in cells, the entire insert was sequenced
I suppose the positive control you mentioned has been detected by the same batch of anti-Flag antibody. As you detected nothing in different vectors and cell lines, the most parsimonious hypothesis is that there is something wrong with the insert. It the FLAG tag is NH2 terminal, did you remove the ATG of the insert? There is sometimes a high bias in the start codon usage when the two are nearby. If the FLAG is C-terminal, be sure that the transcription factor is not trimmed in any way so that the tag is lost. Last can you express the insert in bacteria to check it?
Philippe Fort Thanks for your response I have a question regarding how to determine if the protein was trimmed? How would you determine if your protein is trimmed cause mine is a tag fused within c terminal and the tag is one the vector for my regular transfection, it's the same as my multiple positive controls( we do have multiple genes cloned into the same backbone, some of them even bigger than this gene). And initially, we do think something wrong with the insert, however, I did check the sequences, nothing seems wrong.
By the way, I have a bacterial version of this protein, it can be purified.
Juste ensure that the stop codon has been removed from the insert to get the tag translated. If it's ok, try to put a N-ter tag to your insert. If you have a bacterial version, this means that the insert is fine. Did you use this very one for expression in mammals? Last, although unlikely, the protein might have a rapid turnover (treat cells with MG132 to stabilize) or cells expressing the factor may die or detach (you may reduce the period of recovery after transfection).
Philippe Fort Thanks so much for your comments. So the bacterial construct is specific for Eco.li. It cannot be used for mamalian cell line expression. Regarding your comments why do you think protein degradation is unlikely?
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