Hi Navin, when performing transfection there are numerous parameters involved, cells, cell culture medium, serum, DNA, transfection reagents that it is difficult to have consistent results. For example cells passage number is critical as well as cell growth. Serum composition as well (do you use same serum lot) etc; I can recommend is to try to standardize your transfection parameters, transfect cells always with freshly thawed cells, use the same serum, use the same DNA (ensure your DNA has always the same purity and quality). As for the reagent, company should provide reproducible reagent, for your cells I recommend DreamFect Gold transfection reagent.

Hi there. We usually transfect with polyethylenimine, 6 hours after plating. Using this method you do not have to remove FBS when transfecting, so cell viability remains high, with a high level of transfection. We typically use 1.5 microg DNA per 10 cm-plate, and observe robust expresion of plasma membrane protein after 16-20 hours.

Thanks for your answer. I think there is no problem with transfection because every marker protein expresses well. Although, I have already checked different plasmid concentration , different transfecting agent concentration, temperature effect, different time point after transfection, sodium selenite suppliment treatment time point and its concentration , plasmid purity but in every cases marker protein expresses well , only there is problem in expression of selenoprotein.

Hi I just took a look into this and it seems your problem is related to the cellular concentration of selenium in the form of H2Se. Without the selenide inside the cells, they will not give you your surface protein of interest. Do a literature search and find ways to increase selenium uptake and you should see better results potentially with lines you have already created since you are saying all went well with your markers. I didn't dig too much, but the paper below looks promising. Though it is focused on freshwater plankton, the theories should hold as a guideline for medium parameters to find what your cells do best with.

Riedel, Gerhardt F., and James G. Sanders. "The influence of pH and media composition on the uptake of inorganic selenium by Chlamydomonas reinhardtii." Environmental toxicology and chemistry 15.9 (1996): 1577-1583.

This may not be the best answer, but, if what you need is more protein, just use more plates. I often have to transfect around 6 plates/experiment in order to have enough membrane protein for biochemical analysis. It seems like all this time that you are spending trying to optimize a process that has inherent variation could probably be put to better use by just "biting the bullet" and use more plates, in the long run it may save you time.

Did you used the PLAT-E cells for viral medium collection? and what was the confluency of the cells at the time of transfection with your plasmid? Usually when you transfect the PLAT-E cells, it is better to have below than 70% confluency so you can easily get your desired viral medium in a quite good quantity and then follow the onward steps. If you transfect the PlAT-E cells with greater confluency, it may not produce enough viral proteins containing your codon.

There is no problem with transfection. I think there might be problem with selenocysteine translation and/or transportation of protein to cell membrane. Thus looking for suggestions.

I would agree with John Wokuluk ; I would add Selenium and also I would try to modify the reducing power of the medium