Posted 29 January 2009 - 04:02 AM
Please can someone help
I want to express a protein in HEK293 cells via transfection in order to do functional studies (not for purification etc) so I dont think I want it tagged etc etc. But do you use the full length cDNA or the ORF clone? Both are available. I have no clue - please help!!
I am just interested in maximal expression of the active protein and will assay the cells directly for function.
Thanks in advance!
Posted 01 February 2009 - 08:18 AM
Posted 03 February 2009 - 08:44 AM
Remember that transient transfection will result in like a 1000x fold overexpression. It might not ideal for funcional studies. Whereas making a stably expressing cells where the levels of the protein are not highly overexpressed might be useful. It entirely depends on the question you are addressing.
Thanks for your replies. Yes the aim is to get as much expressed as possible - its really just to screen for novel substrates of that protein, and overexpression has its advantages in terms of detection of uptake into the cell etc.
Hope you dont mind me asking again (not a molecular biologist by training!) but am I right in going for the full length cDNA (not the ORF) as I dont want any modifications to the sequence (purification tags etc). Or am I risking having UTR sequences that might suppress expression if going for the cDNA? It will be in a CMV-promoter driven vector.
Thanks ever so much