My problem:
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 didnUt 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 westernUs 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