Purifying and Concentrating ATP

Bhadresh Rami brami at stanford.edu
Mon Jul 21 21:48:53 EST 2003


Hello Folks,

	I am trying to purify ATP (commercially supplied - Sigma/
Calbiochem) from its hydrolysis product ADP (even 3-5% ADP contamination
is not acceptable for my experiments).

	Running a KCl gradient on an ion-exchange column separates ADP
from ATP.  However, the ATP gets very diluted coming off the column.
I cannot increase the stock ATP that I load on the column, because the ADP
and ATP peaks are closely spaced and at high ATP concentrations, the two
peaks begin to merge.

	Moreover, the ATP elutes at about 50-100 mM KCl.

	My main problem is to remove the KCl from the ATP solution without
enhancing ATP hydrolysis in any manner AND also concentrate the ATP soln.
to obtain a greater working concentration (although the latter is not
absolutely essential).

	changing pH conditions, temperature, lyophilization, etc., could
promote hydrolysis of ATP to give ADP, which defeats the purpose of my
purification procedure.

	either I am missing something very obvious, or it is indeed
difficult to do this!

	Any useful and tested insights on how to purify and concentrate
ATP without promoting hydrolysis will be of great help.

	Thanks.

							Best wishes

						       Bhadresh Rami




More information about the Proteins mailing list