L. Lintott wrote:
> Hi all,
> We are soon going to start doing some 2D work. I personally haven't done
> any 2D work so I was looking for a bit of advice. I am considering the
> PROTEAN IEF from BioRad. I have always been resonably happy with the
> protein apparatuses from this company. Any one using this system and
> happy or not happy with it?
> Also, we want to fluorescently stain and will be imaging with the new
> Typhoon from Amersham. Any suggestions for the best stains to use?
Under all circumstances, get a horizontal IEF with precast gels with
immobilised pH-gradients. Those are offered several companies, iirc also
from BioRad. Personally, I have used Pharmacia's IGPhor system, with
good results (no affiliation).
The old method of tube gels has several disadvantages:
- Samples are added at the acidic end in dilute phosphoric acid. Many
proteins are insoluble under these conditions and do not enter the gel.
- Gels are easily distorted when removing them from the tube, adding to
variability between runs. Remember that IEF-gels need to be very low
percentage and are correspondingly soft. Precast gels are backed with a
plastic film, which makes handling a lot easier.
- Sample size is much more limited in tubes than with the horizontal
system, where you rehydrate the gel with your protein solution. That
increases sensitivity.
The only problem with precast gels is their price, once you have bought
a system from a particular company, you are stuck with their gels and
they know it.
