luciferase assay for ATP

[Mike Kirby]

In article <v01530504ad3e4b3cd15b@[205.232.67.212]>, ryan at hws.edu (Jim
Ryan) wrote:

> Is any one familiar with the use of luciferase for the detection of
> intracellular ATP in cells? 

> Dr. James M. Ryan                       Ph: 315-781-3601
> Biology Department                      FAX: 315-781-3587
> Hobart and William Smith Colleges
> Geneva, NY  14456                       ryan at hws.edu

Dr. Ryan,

A kit is available from Sigma Chemicals (St. Louis, MO).  The basic
procedure involves running an ATP standard dilution series, which must be
constructed each assay day, versus your unknowns.  Care must be taken with
the reagents to limit bacterial contamination.  Also, the reagents are
unstable after dilution from lyophilized powders (a kit is good for about
2 wks, maybe more if you aliquot the material and -70°C store it).  Kits
will run you a little under $200.  This technique cannot measure chelated
ATP (i.e., ATP-Mg).  Measurement usually is performed in a scintillation
counter, with windows set to full spectrum, using HCl-cleaned plastic and
glassware (must be ATPase free; this includes bacterial sources).  The
full protocol is outlined in the Sigma technical supplement sent with the
kit.

-- 
Mike Kirby