problems with transient transfections - (Apr/25/2007 )

hi everyone!!!
so the spring is here, the fields blooms, but my transient transfections still didn't work.
my cell lines, derived form melanoma and ewing sarcoma, are impossible to transfect with my plasmids (taht they work in other cell lines like 293T or HT1080) I have optimized the protocol with the aim of a gfp reporter plasmid, that works!!!, but when I jump to the plasmid that contains the interest protein, nothing happens, when I check for the expression, I find a complete white western, ansd the positive controls works!!!. I checked the plasmids in agarose, and I observed no supercoiled forms, but the same happens with aliquots that works in other cell lines, weird weird. could it interfere the presense of some endotoxins? I would appreciate any suggestions
thanks

-toni towers-

QUOTE (toni towers @ Apr 25 2007, 09:04 PM)

I think the spring or flowers do not appear to be responsible for failed transfection

do you observe any differences of transfected cells in rates of apoptosis, necrosis, viability or cell cycle?

I live with the idea that there are some proteins which are harmful in overexpressed state and would perturb proliferation so that they are restricted in expression; or if their expression cannot be restricted, more cells are killed;

I would be very appreciated if someone could approve my assumption that transfected but harmful proteins will not be expressed or less expressed compared to harmless proteins (such as GFP alone)

-The Bearer-

hi,
I observed the same rate in cell death that also appears transfecting the gfp plasmid, and, I don't think that this protein is a toxic protein for this cells, mainly because they already espress it. I'm tagging it with the transfection. But I agree that the expression overexpression of certain proteins in cells will disturb cell viability, of course ,
Don't you think that polinization could interfere with lipofilic charges?

-toni towers-