Re: Dupont Polyscreen
Hi, all,
I have received some samples for N-term seq analysis that were
electroblotted to Dupont Polyscreen. Some earlier attempts at N-term seq
analysis for this lab have failed and, though this PVDF membrane lists
N-terminal sequence analysis as one of the uses for the membrane, I would
like to know what sort of success other labs have had with this membrane.
A search of the ABRF web site showed three messages with concerns about
using the membrane for Edman degradation--
One message from James Farmer was as follows
"A researcher here has been submitting samples to me blotted on Dupont/NEN
Polyscreen PVDF (P/N NEF1000). She thinks she has placed plenty of
digested protein (thus having a free amino terminus) in her gels and the
Coomassie stain of a gel looks that way.
When she blots the non-stained gel to the aforementioned pvdf, she gets
what I call a "foveal" band (the kind you can only see when you don't look
directly at the band (an old astronomy trick)). I get only a few
background amino acids (1 pmol or less) when I sequence.
Has anyone used this pvdf with success? Has anyone noticed any problems
with it?
Thank you for your ideas and help."
And in response, Mike Scawen in the UK responded (see
http://www.abrf.org/archives/hmail/9820/0013.html)
James
If it is of any help to you I have been sequencing proteins blotted
onto PVDF from DuPont/NEN. The blots were not done by my lab, and I
had assumed that they were on Problot PVDF. The proteins sequenced
perfectly well, but I have come to the conclusion that there is
something in the NEN material that is not present in Problot, as we
have been having some strange effects which we have finally decided
must be due to the membrane, as everything else checks out. This
problem had us and ABI stumped for several weeks.
We see
occasional 'blank' injections, and the transfer line to the 120
becomes full of tiny bubbles, which effectively stop any liquid flow. A
deposit also builds up in the
conversion flask, both on the glass and round the delivery lines. I think
that some component of the membrane is
extracted and builds up in the flask and line. It also appears to
cause low recoveries of PTH amino acids.
So far we have not found
any truly satisfactory way of removing it. Our ABI engineer suggested
methanol, but it did no good. We have tried acetonitrile, which
seemed a better solvent. The only complete solution was to replace
the conversion flask and transfer line, which brought performance
back to normal.
The take home message is that not all PVDFs are the same.
The last message was from David Reim
(see http://www.abrf.org/archives/hmail/9825/0093.html)
Dear All:
I recalled a problem that was described using the DuPont/NEN Polyscreen
PVDF membrane on this bulletin board back in June of this year and
thankfully was already alerted to a potential problem when a researcher
brought his sample to me on this membrane. The researcher assured me that
someone at the Dupont customer service 800 number told him that this
membrane was OK to use in Edman sequencers.
Part of my preparation prior to sequencing involves a brief wash of the
sample bearing membrane(s) with methanol and I noticed that several of the
membrane strips appeared to be melted together after the methanol
treatment
(very curious behavior for PVDF). At this point I called Dupont customer
service and was informed that while the Polyscreen PVDF is OK to use in
Edman sequencers, a recent lot was defective - lot #245174. I the cut a
small corner from the sample bearing membrane and submersed the piece in
methanol. After 10-15 seconds, bubbles where observed coming out of the
PVDF piece. After 3-5 minutes, I removed the piece, dried it with argon
and lo and behold what I was holding appeared to be some sort of matting.
I could see through the piece and there was an obvious weave to it (also
curious behavior for the PVDF membranes I've used). Bottom line is that I
am very grateful to all who contribute to this bulletin board for all the
tips, insights, etc. that I have picked up and hope that this experience
of
mine will help prevent a potential problem for someone else.
Dave Reim
The Wistar Institute
Protein Sequencing Facility
Thanks for any input,
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403
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