supplier of pure ADP?

Kyle Legate legatek at mcmail.cis.mcmaster.ca
Sat Oct 19 10:37:46 EST 2002


On Fri, 18 Oct 2002, Han Broekman wrote:

> Dr Engelbert Buxbaum <engelbert_buxbaum at web.de> wrote in
> news:3DAFC2F6.5C68BD34 at web.de:
>
> > Kyle Legate wrote:
> >> It is common in enzymology circles that commercial substrates are
> >> further purified in the lab before they are used in the assay. This
> >> should be readily accomplished by HPLC with a cation exchange column.
> >
> > In my experience, ion pairing chromatography with triethylamine
> > bicarbonate on a C18 column works better. This buffering substance is
> > very volatile and can be removed by lyophilisation.
>
> May I ask for a copy of the protocol you use to accomplish the
> purification of ADp and/or ATP?
>
Interesting, I use the same buffer system, and a quaternary amine column
(wide-pore QUAT 5um from J.T. Baker). In brief:
Prepare a 0.5M triethylamine buffer (TEAB) and blow CO2 gas through it for
a
couple of hours (I use a flask of dry ice connected to a gas diffuser
stone). Filter it and keep it for up to a week. Equilibrate the column and
load the nucleotide with 4% TEAB, 96% water. To elute the nucleotide,
gradually ramp the TEAB to 100%. Monophosphates elute first, followed by
diphosphates and finally triphosphates.

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legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com

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