mcodevilla at gemabiotech.com wrote:
>> Hello,
> I'm running 2D Electrophoresis and after Silver staining the gels, I
> observed few proteins.
> The problem is that proteins do not transfer from the strip to the gel
> during second dimension, they stay in the strip.
> I try longer equilibration times and other IEF methods without success.
> Does anybody know how to solve this problem?
What kind of equipment are you using? Have you stained your first
dimension gels and verified separation?
I had no ends of problems with 1st dimension gels where the sample is
added at the acidic end, dissolved in phosphoric acid. Under these
conditions most protein never enters the gel. However, with modern
horizontal gels, using immobilised gradients, I had good experience. I
used IGPhor from Pharmacia (no affiliation), but similar equipment is
available from several other companies.
Once your 1st dimension gels are ok, make sure you equilibrate it with
sample buffer which contains reducing agent (DTT or BME) and detergent
(usually SDS) for about 20-30 min on a shaker. A little bit of marker
dye (bromophenol blue) helps to verify that the sample buffer has
completely penetrated the gel. Don't use to much, the solution should be
transparent water-blue, rather than the midnight-blue that most receipes
call for.
Then mount the gel, making sure that there are no air bubbles trapped
between your 1st dimension gel and the top of the 2nd dimension gel.
This is crucial, as you can squeeze a tiny air bubble into a thin,
virtually invisible insulating layer between the gels. Glue it in
position with 0.5% agar in stacking gel buffer, working quickly as the
stuff hardens immediately.
If you still have problems, check that the proteins have left the 1st
dimension gel by staining it. Is the polarity of your power supply
correct (stupid question, I know, but...)? Do your proteins stain well
with your receipe if you run them directly on a Laemli gel?
Without further info on your setup, this is all I can think of. There is
a nice free booklet on IEF-techniques available from Pharmacia.
