Recently I generated stable SP2/0 cells carrying my gene of interest. I used pcDNA6/v5 His A as a vector and blasticidin S as a selective antibiotic. The vector was delivered into cells by electroporation.
The cells are clonal and resistant to 10 mg/ml of blasticidine S. I proved protein expression in Dot-test using HisProbe. Cells were harvested, washed by PBS and mixed with 3 M urea in order to expose His-Tag. Final cell concentration was 10^5/ul. All cell clones were positive and intact cells were negative. But when I stained these cells by specific monoclonal antibodies to the protein of interest and put them into a flow cytometer it turned out only one clone had a small positive peak very close to the negative one and other clones looked identical to intact SP2/0.
What's wrong with them? Could anyone help?
Low copy number of the gene of interest could be one explanation. If so, could an additional cycle (or cycles) of transfection help?
Thanks.
Dear Ilya, the first way is the use of a plasmid with strong promoter. Sometimes the use of a single plasmid expression could varies between cell types. Another good strategy is the coexpression of your gene and one Chaperone at the same expression system, perhaps using two plasmids. Chaperones stabilizes the protein expression and folding. Please check some examples here http://www.sciencedirect.com/science/article/pii/S0167779910000314. You probably need to check some collateral effects of this aproach here: http://www.biomedcentral.com/content/pdf/1475-2859-9-64.pdf. Good Luck.
Other example http://onlinelibrary.wiley.com/doi/10.1002/btpr.192/abstract;jsessionid=8307EC7985A7F8D426B9AEC5A8A80A80.f04t01?deniedAccessCustomisedMessage=&userIsAuthenticated=false
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