Dear,

I had a trouble in making stable protein expressed cell.

I made a expression vector with quite long (> 1000 aa) in fusion with selection marker (Zeocin) resistant gene. After transfection, the result of immunofluorescence assay was very good with bright signal of about 20% of the stained cells.

After 2 weeks of selection with 100 ug/ml Zeocin, the non-transsfected cell were all died and we got resistant colonies (very few) in transfected cell population. However, immunofluorescence assay of the cells from that colonies showed no signal or very low comparing to the control (non-transsfected). We did several times and the results were still the same. Then, we did PCR for the target gene and we got clear band for the presence of the gene in transfected cells. Note that Zeocin was still in the culture medium when we did immunofluorescence assay.

I considers that whether Zeocin weaken protein expressed from the transfected gene and should be removed before immunofluorescence assay for better result.

Thank you very much.