as far as I recall the first time this flow-through approach was described
in detail was in 1993 by T. Tetaz ,E. Bozas,J. Kanellos,I. Walker and I.
Smith in Techniques in Protein Chemistry (ed the famous R. Hogue Angeletti)
Vol IV, pages 389-397, Title: In situ tryptic digestion of proteins
separated by SDS PAGE: improved procedures for extraction of peptides prior
to microsequencing.
We used this method, which involves the use of an empty small HPLC column,
for a number of years and it worked quite nicely. We switched to the
conventional digestion in tubes because the flow-through method was not
really ammenable to multiple proteins being processed at one time (it's
easier to handle a dozen tubes at a time, a bit more tricky to open and
close a dozen LC columns) and solvent changes (involving HPLC pumps) were a
bit more tedious than pipetting.
That said, that was five years ago now and the thought of automation of the
digestion process involving a miniaturized flow-through system has occurred
to me more than once over the intervening years, I hope Gary is developing
something along these lines?
Regards...Ken
>I heard that Gary Hathaway has been using or experimenting with an in-line
>cartridge into which the gel pieces + enzyme are placed, then the digest
>liquid is filtered through to the microbore column. If he and/or anyone else
>has experience with this, I would be very interested in more information.
>Thank you very much.
>Dr. Meredith Gould
>Facultad de Ciencias
>Universidad Autonoma de Baja California
>A.P. 2921, Ensenada 22800, B.C. Mexico
>
>tel/fax 011-52-61-744-560
>mgould@bahia.ens.uabc.mx
********************************
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.medstv.unimelb.edu.au/abrf.html
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