Koch Lab:Protocols/Kinesin/ATP suspension

The book, Molecular Cloning, says to suspend ATP in a Tris buffer at pH 8.0. Cytoskeleton has conflicting protocols that say to either store in Tris at pH 8.0 or in DI water. The reason why we want to store ATP in a buffer that has a high pH is because it auto-hydrolyzes less in alkaline buffers as opposed to acidic ones. This paper talks about ATP hydrolysis in different buffers and shows that auto-hydrolysis doesn't change very much for ATP stored in buffers at pH 7.

In order to prevent as much auto-hydrolysis as possible, since our PEM buffer is at a pH of 6.9, it is a good idea to keep the ATP at -20˚C at all times. It is also a good idea to aliquot it into convenient amounts so that large stocks do not have to be kept out of the freezer for more time than is necessary.

The ATP should be put into our PEM solution at a concentration of 100 mM. It should then be aliquoted, flash frozen and put immediately into a freezer storage box which is then stored in the -20˚C freezer. You can use one of the blue freezer boxes to hold your stock solution while you aliquot to prevent the stock from being at room temperature too long.

The ATP we use is can be found |SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC here. It is a magnesium salt so it will go into solution very easily.



Make sure you label the tubes before putting them into the freezer.