Help: low transfection efficiency but control works great

Ian A. York iayork at panix.com
Thu Jun 1 21:19:10 EST 2006


In article <mailman.60.1149185411.18505.methods at net.bio.net>,
Yulia Shifrin <yulia24 at gmail.com> wrote:
> Hello everybody!
>I'm having problems with transfecting melanoma cells with vector containing
>dsRed fused to my protein of interest. I'm using Fugene 6 and get 60-70%
>efficiency with vector-only control, but only 1-5% efficiency with my
>construct. The protein that I'm working on is pretty large (300 kDa). I'm
>getting really frustrated. Has anybody encountered similar problems? What
>can i do to improve the efficiency

A couple of simple things that you've probably already thought of -

(1) Have you repeated the vector prep?  Different maxipreps, even using 
the same system, can give widely different efficiencies; that's the case 
even when the two preps seem to be the same in concentration and quality.

(2) How stable is the protein you're interested in?  If it's degraded 
rapidly, then the dsRed will also be degraded along with it and of course 
the intensity will be much, much lower.  If you have antibodies to the 
protein you can immunoprecipitate (not western blot) using a short label 
to detect expression.  I believe there are antibodies to dsRed as well, so 
you could IP with that as well.

Good luck.

Ian 


-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England


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