protein expression problem

Ricky LEUNG b883732 at logic.itsc.cuhk.edu.hk
Mon May 28 09:23:36 EST 2001


Hi everyone,

I have some difficulties to express my protein in COS-7 cells. 

I have cloned the insert into pCDNA3.1 vector and checked the reading
frame by sequencing. Then I transfected it into COS-7 cells by Lipofectamine
Plus and performed stable transfection with 750 ug/ml G418
selection. After 4 to 5 weeks, I harvested the cells and did the western
blot using Anti-His Ab from Santa Cruz. No band was detected. On the other
hand, I extracted total RNA from the transfected COS-7 cells and did the
RT-PCR. The insert was over-amplified when compared with the control
vector without insert. So, what is the problem? No protein was found. Is
it related to translation problem? How can I do?

Any comments are welcome.

Thanks in advance

Ricky




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