>A customer brought me a Coo-blue stained blot with a faint but visible
band on
>it to sequence. I put it on my sequncer and got no sequence. I told
the
>customer that either there was less protein in the band than we
thought or it
>was N-terminally blocked.
>
>We sequenced again with (she tells me) more sample -- still no
sequence. We
>decided that it must be blocked and decided to digest it.
>
>The expected protein has plenty of tryptic sites so we red/alk and
then did a
>tryptic digest(on PVDF). (DTT/Iodoacetate, Promega trypsin) I ran
the digest
>on a sample slice and a blank slice (of PVDF) in parallel. When I ran
the
>digests out on my microcapillary HPLC, the digest of the sample PVDF
looked
>exactly the same as the digest of the blank lane. I have to conclude
that the
>protein didn't digest, but why ?
>
>At this point my customer only has a little bit of the sample left (we
only
>get 1 more shot). She needs enough sequence to prove that the protein
is what
>her western's have led her to believe it is.
>
>My questions:
>
>1) Are there proteins out there that are Trypsin resistant even
after
>reduction/alkylation ?
>
>2) What other possible explanations are there ?
>
>3) What would you do next ? (Our only idea is to try CNBr?)
>
>Thank you all for all the past help and thanks in advance for help
with this
>problem.
>
>Weather Inconsequential(Charleston S.C.):
>Spring has sprung and if you can avoid sinking up to your ankles in
mud, the
>azaelas are beautiful !
>
>Rebecca P. Ettling
>Biotechnology Resource Laboratory
>Protein Sequencing and Peptide Synthesis Facility
>Department of Biochemistry (Rm 505)
>Medical University of South Carolina
>171 Ashley Avenue,
>Charleston, SC 29403
>Tel.: (803) 792-1271
>Fax: (803) 792-1264
Gautam Sarath
N-226, Beadle center
Protein Core Facility
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842