I am curious about your comment..."When I ran the digests out on my
microcapillary HPLC, the digest of the sample PVDF looked exactly the sam=
e
as the digest of the blank lane."=20
Does this mean that there were peaks generated in the digest of a blank
piece of PVDF or were they both completely blank? Wasn't there a peak
corresponding to the undigested protein in the sample digest, or did it
disappear?=20
regards, Sandy Kielland
At 09:36 AM 3/2/98 -0500, Rebecca Ettling wrote:
>Hi Everybody,
>
>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 th=
e
>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 t=
he
>digests out on my microcapillary HPLC, the digest of the sample PVDF loo=
ked
>exactly the same as the digest of the blank lane. I have to conclude th=
at
the
>protein didn=D5t digest, but why ?
>
>At this point my customer only has a little bit of the sample left (we o=
nly
>get 1 more shot). She needs enough sequence to prove that the protein i=
s
what
>her western=D5s 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
>
>
>
>
>
>
>