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.