Hi, all,
Here is, I think, a word to the wise.
I had a lousy experience with PVDF last week that I want to relay. I
would not have had this problem if I routinely tested samples that I
received with Len Packman's suggested procedure of dunking the PVDF into
methanol (containing 0.1% triethylamine). Obviously, samples that have
been through the stain and destain procedures used for Coomassie Blue
will sort out the PVDF from nitrocellulose long before you get the
sample for protein sequencing--but beware those pieces that are not
blue.
I received a sample of 6 "PVDF" pieces--called to ask why they did not
have any dye on them, as I wanted to trim them down to fit into the
Procise cartridge--he'd run a separate gel and cut out where the band
had run--it was his money so I loaded them on the sequencer, set it for
8 amino acids and went away. I returned to my horror to see a cloudy
flask, kinda wispy looking stuff, with wisps of material hanging off of
the pickup tube--and of course sensor errors for loading the sample
loop.
Opened up the reaction cartridge, and those 6 pieces of PVDF were, of
course, down to a high glossy yellow spot on the teflon seal.
And what a distinctive series of chromatograms for the amino acids at
each cycle--I'll be glad to forward to anyone the Procise 610 file of
the chromatograms so you too can recognize nitrocellulose peaks.
Luckily, I had a control sample of the Biorad Immunoblot PVDF from the
(almost now x-) "client" and tested it in the sequencer chemicals--off
machine of course! Ethyl acetate does such a beautiful job of
solubilizing the stuff. Where was my sense of humor.
After two phone calls out to the world--Dick Cook and Joe Fernandez--I
got advice to replace what lines you could and the flask--I did that and
washed, soaked, washed, soaked in ethyl acetate for an extended period
of time. Getting pretty adept at manually moving solvents around the
machine.
Today, the sequencer (knock on wood--well, throw out the nitrocellulose)
looks like it is running just fine. But this could have been a very
expensive problem--I'm glad I didn't run their control sample back to
back with the unknown--and that I didn't have 4 carts to load up and
leave for the weekend to really clog the whole thing up.
In addition, it is great that the Procise does deliver so much ethyl
acetate during the runs--there is on the one hand the problem that it
moves the nitrocellulose so well from the reaction cartridge on down the
lines, but on the other hand, it does move it so well--hopefully I don't
have residue sitting anywhere to haunt me later.
It's a challenge, too, dealing with a post doc who is adamant that they
used PVDF--so you call Biorad tech support to see if that lot number had
any problem--and, by the way, Biorad tech support was incredibly
responsive--they wanted me to test more of the clients PVDF with
chemicals and sent me a batch to compare side by side. Bottom line,
Biorad Immunoblot PVDF works just fine.
This morning the post doc sent through this message to me
> I have come to the conclusion that it is likely that I erred and sent you
> nitrocellulose in lieu of PVDF; I cut the piece I left for you from the roll
> but I had taken the sheet I blotted from some loose pieces in the PVDF tube.
> The few loose pieces still there are a mixture of PVDF and nitrocellulose so
> I must have blotted the nitrocellulose and sent it to you... This would have
> been just a dumb mistake but I should have caught it when I wetted the PVDF
> with MeOH, it was late and I was tired and I must not have done this. I
> greatly regret damaging your instrument. Had I known switching the membranes
> was so serious I would have tested the blot for solvent sensitivity; I
> truthfully thought nitrocellulose simply didn't work, not that it trashed
> your sequencer. Is there anything to be done to clear it out?
>
I like the part about the few loose pieces being a mix of nitrocellulose
and PVDF. Can happen at any time in any lab. So, any non-blue PVDF
from now on gets tested before it goes on our trusty Procise---and
before I am so trusting of blue, are there any blue dyes used on
nitrocellulose that don't involve wetting in methanol?
Now, on to the next thing,
Deb McMillen
Institute of Molecular Biology
University of Oregon
Eugene OR 97403
This archive was generated by hypermail 2b29 : Thu May 18 2000 - 09:47:34 EDT