>Proteomics is taking off with Genomics Solutions, Pharmacia, Micromass, PE
>etc offering 'complete packges' anywhere from gel running through to ESItof
>(some packages real, some due for 2000 launch). Central to the successful
>operation of these setups must be an automated in-gel digestion system
>whose ability to deliver on low abundance samples must match or exceed that
>achievable through 'dirty' manual means.
>
>My question: who has experience of automated digesters and possibly
>automated clean-up devices/spotters? Are they any good? Are they useful at
>the sensitivity needed to identify Ag-stained spots?
We have been using a Canberra-Packard Robot to perform tryptic digests for
some time now and we are quite happy with it's performance. We are
examining the newer model as the current one has a few age related
problems. At present, it is not suited for ZipTip clean-up but the
manufacturers have assured us it could be modified for such a purpose.
As far as sensitivity goes, we find it really depends on the peptides. I
have had success with Ag stained spots that are digested and spotted onto
the MALDI target using the robot. By the same token, some light CBB spots
do not work. Generally, we find the system works well down to at least
1pmole loaded onto the gel (it will go lower but to "guarantee" results I
suggest 1pmole as a minimum). Our general approach is to PMF all spots,
those that are not identified we would then use ZipTip clean-up and finally
if still no identification then MS/MS.
That's the short version. If you want more information just let me know.
Hope this helps,
Peter.
I'm afraid I don't have a clever saying to put here.
Peter Hains (PhD) Ph. +61 2 9850 6216
Australian Proteome Analysis Facility Fax. +61 2 9850 6200
Level 4 Building F7B E-mail. phains@proteome.org.au
Macquarie University, Sydney 2109 Web. www.proteome.org.au