banging my head against the wall - (Jan/27/2006 )

I have been growing clones of a pbmn-ires-neo vector with a flag tagged 16kD protein insert in T47D cells. I have established two stable lines one through transfection and one using a retrovirus. These lines are growing well in the Neomycin spiked media at the same concentration that killed off all the other cells. The problem is I can't pick up the protein expression on Western blot. The + controls do come through. ( I have been going for the flag tag). Have varied the lysis buffer, the detection systems, immunopurified... Is it possible that my cells could have incorporated the neo resistance gene without the rest of the plasmid?

thx

this might be a dumb question, but have you sequenced the plasmid? perhaps there was an issue with frame or something on the tag and it is not expressing properly, even though the plasmid has been incorporated...

I agree with aimikins, did you checked if its on the right orientation? I am precisely in the same problem, I have stable cell lines selected on puromycicn but cant find my protein in westerns, so I am sending my insert for sequenciation to verify orientation

I just realized that I am out of date hahahah

No.. it was posted yesterday.... nevermind....check orientation or look expression at RNA level. am also doing that and trying to see if my protein is not retained intracellularly, I wabnt to find it in conditioned media

Thanks,
I am working with 2 cell lines grown from 2 separate plasmids - one we have not been able to sequence and one we have - which is correct. neither are expressing.

for genomic integration that is not an accurate question regarding the retrovirus mediated delivery.
For other one, you pmay have the possibility of a bad opening of the plasmid but i don't think so.

you may have to increase the neo concentration. And are the non transfected cells all died?
to check presence of your fragment of interest you may do a PCR on genomic DNA.

Almost all of the cells died - except the clones that I grew my cells from. I agree - I probably do need to sequence....