Nucleotide Mix Preparation

Materials

100 mM Mg·ATP

gamma-³²PATP or alpha-³²PATP

DDW

10 mM MgCl₂

Procedure

1.	Prepare mix by mixing the following materials:
	10 µL 5 µL 80 µL 5 µL ^______ 100 µL	100 mM Mg·ATP gamma-³²PATP or alpha-³²PATP DDW 10 mM MgCl₂

Notes

1.	The amount of ³²PATP added depends on the specific activity and should be adjusted to give about 0.5 µCi/µL = ~10⁶ cpm/µL.

2.	Gamma-³²PATP is used to test for nucleotide hydrolysis and alpha-³²PATP is used to test for nucleotide binding and hydrolysis.

2.	The amount of unlabelled Mg·ATP in tests with alpha-³²PATP can be reduced to give final concentrations of 1 µM, 3 µM, 10 µM and 100 µM in the reaction mix. These reactions can be used to determine the K_m for nucleotide binding and hydrolysis.

If you use methods from the Kinesin site, we ask that you cite the Kinesin Home Page and authors, or the appropriate source publication in your work.

Created 8 December 1999 18:52 GMT
Modified 7 May 2001 18:30 GMT