I would be interested in other people's experience with making their own
dry-strips.
The two most difficult parts seem to be rehydration of the dry strip in a
sample and getting good transfer of proteins from the dry strip to the
second dimension. Relatively high molecular weight proteins are very
problematic (above 120 kDa) and we have better luck with them simply using
a 4-8% long SDS-PAGE gel (not doing the focusing)--there are so few
proteins in that mass range that they can usually be separated from one
another on a 1 D gel.
Transfer of a 2D gel to a Western blot is more difficult than you will
anticipate because the gel is thicker than the normal one-dimensional gel,
the gel has a greater tendency to buckle in the transfer buffer. Plate
electrodes give better results, but the bands still tend to look weird. We
have to stain the Western blot before doing the antibody reaction in order
to orient one gel to the other.
You may need to use the zoom gel approach and basic gels to get complete
coverage. To get minor bands you need to swell the dry strip with the
sample and/or use zoom gels. The problem with fractionating the sample is
then getting the sample into the buffer required for the swelling. We've
seen protein loses in this stage.
Cutting bands out and doing the mass spec is relatively easy. In a human
system, about 75% of the bands can be identified by maldi. For weak bands,
we have no problems with amounts using LC/MS on an LCQ, with multiple users
who don't want to become mass spec experts. For these low intensity bands,
we normally run 4-5 gels and cut out the spots and pool them for analysis.
Nanospray from a needle is relatively difficult to do. We also got that
set up and working, but it took about five months and only one of four
people who were using it got reasonably good at working below the 5
picomole level. And it always seems as if the most precious sample is the
one where the needle clogs.
Katheryn Resing