Hi Steve,
I have previously had similar experiences to yours, with the
exception that my sequencer is an old Porton 2090E. (Porton was
taken over by Beckman a few years after we acquired our sequencer.)
For that sequencer, the R2 reagent is diisopropylethylamine (DIPEA)
in water and, as you observed, it is sold for an exorbitant price by
the suppliers of sequencing reagents. Sigma has tried to undercut
the sequencer companies by about 10% or so with the sequencing
reagents that they supply, and I have actually used the Sigma
reagents before and found them to be pretty good. Still, their price
for the Porton (Beckman) R2 reagent is $146.80 for a 125 ml bottle of
a 4% solution. On the other hand, their sister company Aldrich sells
redistilled, 99.5% diisopropylethylamine at a price of $28.15 (at
least when I last ordered some) for 100 ml. A few years ago, I spoke
with Henry Krutsch, who first suggested using DIPEA as a replacement
forthe triethylamine that Porton previously used as the base in R2.
He prepared R2 by adding 1.5 ml of DIPEA to 125 ml of H2O; thus
making it a bit more dilute than the 4% solution sold by Sigma. I
typically add about 2 ml to 125 ml of water, and it works perfectly
well. You can easily calculate the difference in cost between the
"home made" and the commercial reagent. I think that, among other
things, you pay a lot for quality control when you buy the commercial
reagents.
One nice thing about Porton was that it was a friendly
company that was willing to tell you exactly what went into each of
the sequencing reagents. I am not sure that ABI is quite so
forthcoming. Still, I think it may be worthwhile for you to try
making up some of your own reagents and test them on standard
proteins, replacing the commercial reagents one by one. I now
prepare all of my own reagents except for PITC, which I buy as a 5%
solution either from ABI or from Sigma and then dilute to 2% with B&J
heptane. (Interestingly, 2% PITC in heptane was shown to be a good
concentration to use by the protein chemists at Sigma, who presented
their results in a poster at a Protein Society meeting a few years
ago.) By saving on the cost of reagents, you will be able to run
more sample proteins for quality control purposes without making
things prohibitively expensive.
Sorry that I can not comment more specifically on the exact
situation concerning N-methylpiperidine. Perhaps someone else will
be able to give more specific advice.
Sincerely,
Dan Brune
Dept. of Chemistry and Biochemistry
Arizona State University
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