I am working on a protein which is encoded by 6.3kb of cDNA. The expected molecular weight of the protein is 236kDa. I have the commercially purchased cDNA clone of the same in a Myc-DDK backbone under a CMV promoter. Upon transfecting HEK293T cells, the protein however does not seem to be expressing. What could be the possible reason for this? Is the vector backbone a problem? (The cNDA sequence appears fine. No frameshifts.)
Andreas
I don't have a GFP tagged to my protein yet but I co-transfect the cells with pMAX-GFP and the transfection efficiency ranges between 60-70%. This does not definitely give me the transfection efficiency of my plasmid of interest and I expect it to be lower keeping in mind the size (but the control sets the probability of transfection of my plasmid).
I have antibody against my protein as well as flag. And I have tried western using both. But none picks up the band. Flag gives too many non-specific bands and they are all the same in EV, UT and WT. With my antibody I am able to pick up a single band at a lower range than expected but again it looks the same in the transfected and untransfected.
I am not sure if the protein is toxic (I don't think it is) but I don't know the turnover rate of the protein if it might be degrading fast. I am not clear about expression rate. How do you check expression rate? I am trying to check the same at transcript level.
Could you tell us a little more about the protein. Is it a cytosolic protein or a membrane protein? Have you tried immunofluorescence?
As Vinit and I have suggested, try immunofluorescence. It may be that your conditions for running westerns are not conducive to seeing the protein. It's a large protein. Do you have confidence that it is being transferred to the membrane. A little SDS in your transfer buffer and no methanol will help this. Are you heating your proteins in sample buffer before running them? this to may be a problem. Try just leaving in sample buffer at room temperature for 30 mins before running them. Don't panic, there are lots of things you can try.
Hi Shalini,
I think you are trying to express your protein by transient transfection and I also think your vector has a neomycin resistance gene... even the HEK293T cells already have the neo cassette... I like to ask whether you have checked the expression in other normal cell lines like CHO, NIH3T3 cells or use HEK293 not 293T. I was facing similar problem for long time to make stable cell lines and I was using neomycin resistance gene for the selection and I found there are lots of genes' expression alters in the cells stably transfected with neomycin resistance gene .... you can check the following paper....I dnt know how but CMV promoter also affected in the stably transfected cells....
But I will definitely suggest that you may also need the UTRs for efficient expression... good luck :)
Neil
The protein is uncharacterized and is predicted to be cytoplasmic. I have tried immunofluorescence with both the flag and protein specific antibody. It did not give any reliable input. However, I was able to see a nuclear signal with the flag antibody (but I am not sure if it is a true signal). I have tried out westerns with sds but not withut methanol (I can try that). But I have used heated and unheated protein lysates. The result remains the same. Although I have not left it in buffer at RT for long time. I load my samples immediately after preparation.
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