I've optimized the transfection conditions, and I'm getting a very good transfection efficiency as measured with a GFP plasmid. However, western blot and ELISA analysis of the cell lysate 24-48 hours after transfection indicate that the protein's expression is only decreased by about 50% of non-transfected cells.

Is this to be expected when using siRNA to knock down the expression of a transmembrane protein? My understanding is that completely abolishing the protein is not a feasible goal due to the presence of already synthesized protein in the cells' membranes, however, I have been told that the expression should decrease to near 0%?

Would performing two transfections on consecutive days (or separated by a day) be a viable strategy (assuming that it doesn't cause excessive toxicity - under the current protocol the transfection has very little effect on cellular viability)?

Thanks for your input.