do you have any good evidence that cutting the gel into mush makes the
slightest bit of difference with respect to peptide recovery?
We have had many years experience digesting radiolabelled proteins from
gels, mostly for phosphorylation site analysis, and thus have the luxury
(?) of monitoring peptide recovery with a high degree of precision on many
hundreds of digests.
In our experience, providing the labelled tryptic peptide is of a
reasonable size (less than 30 residues), we routinely achieve 93-98%
recovery of the radioactivity with three 30 minute washes on a sonicating
waterbath of the WHOLE digested gel slice.
I have seen lots of in-gel digestion protocols that call for gel mush but
never any evidence to support a manipulation that you correctly point out
is fraught with technical difficulties. I suspect the diffusion rate of a
peptide in a 10% gel, mushed or otherwise, is extremely high and that
people have confused peptide and protein diffusion rates.
Indeed, I have done some limited control experiments with labelled
proteins, using the method of Cohen, S. L. and B. T. Chait (Mass
spectrometry of whole proteins eluted from sodium dodecyl sulfate-
polyacrylamide gel electrophoresis gels." Anal Biochem 247(2):
257-67(1997)). The diffusion rate of whole proteins from gels into formic
acid:isopropanl:water definitely calls for mushing, we use the disposable
microfuge tube pestles for this style of extraction.
It will be interesting to see if anyone else has data to support mushing
prior to digesting?
Weather inconsequential: enjoying a long hot, gopher-free, summer
down-under, still 38 degrees C outside at the moment, I'm off to the beach
for an evening picnic.
Kindest regards....Ken
> We are attempting to get on the ol' proteomics band wagon (wonder if the
>wagon can support the weight)...anyway we have a vexing problem...the problem
>involves cutting spots out of 2D gels, mincing them into wee tiny pieces and
>transferring the wee tiny pieces into the PCR-tubes with wee tiny holes in the
>bottom that are used in the ABIMED DigestPro robot (whee, high tech!).
>Anyway
>what has been worked out is to put the wee gel piece on a ceramic tile and
>then
>using a fresh razor blade (a new one for each slice...Gillette stock just went
>up ten points) mince the slice into "the smaller the better" pieces, one then
>"scoops up" the mush and gets it somehow into the PCR -tube (note here
>that the
>tube has holes in the bottom, so one cannot add the slice to the tube and
>mooch
>it with a pestle like implement). So, what is the problem you ask? While
>cutting into wee tiny pieces, the pieces: 1.) fly all over, 2.) stick to
>the razor blade...in addition, the human doing the cutting goes blind, gets a
>big headache and contaminates the sample with hair and saliva (from muttering
>disgustedly). Any ideas?? (PS. yes my graduate school mentor was from
>Scotland)
>
>Weather inconsequential: last time around I very diplomatically pointed out
>how nice the weather is out here compared to places like, say Omaha...it was
>pointed out to me (also very diplomatically) that at least those individuals
>located in Omaha don't have any gopher/mole problems at the present time...my
>gopher/moles must be pansies because they have left my backyard alone for
>awhile (gone south for the winter?).
>
>Jim Bloom
>Bayer Corp
>Berkeley, CA
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Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA
Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676
Email: k.mitchelhill@medicine.unimelb.edu.au
Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org
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