Hello everyone,
i am trying to overexpress a protein in HEK293T cells. I have cloned it into an expression-vector with a strong CMV promotor and transfected the construct into the cells with high transfection efficiency.
What amount of secreted protein can I expect in the conditioned supernatant 72 hours after transfection? Should it be enough to detect it via coomassie staining?
Greetings
It depends on several factors like type of protein, half-life, rate of secretion, culture conditions etc. Is there any published literature available for the protein?
What you can do is to take the samples every 12 hours and do SDS-PAGE analysis in comparison with culture medium from the untransfected cells.
In addition to my previous answer, I also suggest to check the cell lysate first to check the expression. And also, the protein secreted into the medium may not be detectable by normal coomassi staining. I suggest to concentrating the protein by precipitation or centricons.
I agree with Dr. Budida. You need to optimize the time period by collecting samples (culture supernatants) at different time intervals and check for your protein. If you protein of interest is tagged with any of the sequences like FLAG, HA, c-myc, etc then you may use tag specific antibodies to check via western blotting.
good luck
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