Help: low transfection efficiency but control works great

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
Thanks in advance
Yulia
<[Only registered users see links. ].net>

In article <mailman.60.1149185411.18505.methods@net.bio.net >, "Yulia Shifrin" <yulia24@gmail.com> wrote:

Yulya, welcome to real life

The thing is, no two proteins are the same. And different proteins behave
differently. They have different steady state level of expression (a
balance of transcription/translation and proteolysis/degradation).
What you are describing is *not* proper estimate of transfection
efficiency. You'd be better off co-transfecting two plasmids - one
encoding plain RFP, another your protein. Then the readout of
fluorescence will have more to do with transfection per se (and even then,
it's perfectly possible that your protein influences RFP expression).

If it's of any consolation, I had a case recently expressing two versions
of a ~ 95K protein functional chunk. They differed in 2 kDa, yet shorter
version expressed ~ 10X worse.

- Dima

In article <[Only registered users see links. ].net >,
Yulia Shifrin <[Only registered users see links. ]> wrote:

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 ([Only registered users see links. ]) <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