We complete transient transfections with HEK293 cells. Recently we have seen a dramatic decrease in protein expression with proteins we have expressed times before. We've checked the equipment for defects, the consumables & reagents remain the same, we've sequenced DNA . Any tips?
May be you can do a pilot experiment with GFP, to check the transfection efficieny (compare old and new method/reagent/similar things). And if you still have your plasmid you used previously, check again with old plasmid and new plasmid in parallel to see the plasmid prep is ok. Also check for any contamination around your lab/system.
Hi Mel,
Check that your HEK293 cells are 50-80% confluent when you do the transfection. I have noticed that if my cells get too confluent transfection efficiency can drop from ~70% to as low as ~1-2%. You should also make sure the cells are not reaching 100% confluence during normal passages.
Alex
http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/transfection-protocol/transfecting-plasmid-dna-into-hek-293-cells-using-lipofectamine-ltx-reagent.html
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