in situ digest

Holt, John - PMC-US (JHolt@US.PMC-Vacc.COM)
Mon, 17 Aug 1998 18:17:25 -0400

I would like to use in situ cleavage, (with sequencing and ms) to
identify bands in the10 to 20Kda range. They are well resolved on
Tricine gels, e.g., 10-20% gradient Ready Gel, Biorad. Has anyone
succeeded with gel pieces from such small pore gels? Is this a case
where transfer to PVDF might be strongly preferred as a means of making
the proteins accessible, rather than simply an operator choice, as was
the conclusion of the ABRF study on in situ cleavage??

I have begun the obvious empirical approach using myoglobin and its CNBR
fragments (17, 14, 8, 6, 2.5Kda) but I will greatly appreciate any
advice.

John Holt

Pasteur Merieux Connaught