I have had some experince with membrane proteins where the goal is to
obtain sequence data, and the biggest problem is HPLC. It is best to avoid
HPLC (particularly reverse phase) at all cost. I would suggest in-gel
digestion of the membrane protein with trypsin or Glu-C (it often works
better than in solution), load the gel pieces into a gradient gel for peptides
(4-20%), blot to PVDF (small pore for sequence analysis) and stain with
coomaasie. In general you should get several bands that you can
sequence directly (you can run a test of the digest by running a silver
stained gel on a small portion of the sample).
We had good success obtaining long strecthes of sequence (including
transmembrane sequences) from G-coupled 7-transmembrane receptor
proteins using this method. Good luck and remember that RP-HPLC
doesn't work for everything.
Jeff Hulmes
Senior Scientist
Protein/Synthetic Chemistry
ImClone Systems Inc.
180 Varick Street
New York, NY 10014
(212) 645-1405
Fax: (212)645-2054
JeffH@ImClone.com
>>> "gsarath@unlinfo.unl.edu" 07/15/98 05:35pm >>>
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