supplier of pure ADP?
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
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.
... . . . . . . . . . . . . . . .
legatek at mcmaster.ca Kyle Legate legatek at hotmail.com
Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
. . . . . . . . . . . . . . . ...
More information about the Methods