Tryptic digest -only trypsin peaks

Gautam Sarath (gsarath@unlinfo.unl.edu)
Wed, 15 Jul 1998 11:31:31 -0500 (CDT)

Dear all: HELP, I ran a routine trypsin digest using Promega's modified
trypsin and the ABRF protocol. I collected several peaks, not observed in
the blanks, and only found fragments of trypsin upon sequencing. This has
not occured on any of the large number of earlier samples. I am also
checking with Promega regarding this specific lot number. The sample was
in-gel digest of about 100 picomoles of a transmembrane protein. I do
observe at least 2 to 3 peaks attributable to trypsin, generally eluting in
a specific region of the HPLC chromatograms fo most of the samples I have
analyzed to date.

Questions:

1) Is the trypsin toying with me?

2) Should I switch to Lys-C (Is the Wako product still backordered?)

3) Should I first try a short (12 hr CNBR gas-phase treatment of a gel
slice) then use fresh trypsin?

4) I will also reanalyze the old gel slices to see if the protein was only
partially digested.

Thanks in advance for the help, Gautam

Gautam Sarath
N-226, Beadle Center
Protein Core Facility - Center for Biotechnology &
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842
http://www.biotech.unl.edu/Proteins/index.html