Highly sensitive detection of ATPase activity in native gels.
Native electrophoresis is a powerful tool for the separation of intact protein complexes. By incubating such gels in a suitable reaction solution, specific enzyme activities can be screened comprehensively. The recent standard procedure for determination of ATP hydrolysis activity in blue or clear native gels is based on formation of a lead phosphate precipitate. The resulting white bands are challenging for detection and documentation of low activities. For the analysis of photosynthetic ATP synthases, the method has to be adapted to deregulate the inhibition of latent ATPase functions. Therefore, we introduced an incubation of gels in detergent solution, whereby taurodeoxycholate turned out to be the most efficient activator. In order to detect low ATPase activities, a short additional incubation step subsequent to the formation of lead phosphate is recommended. By adding ammonium sulfide, the white bands are converted into brownish-black bands of lead sulfide. Our new procedure sustains the linear quantitation range of the original lead phosphate protocol and moreover expands the detection limit.
Suhai
Tina
T
Physical Biochemistry, Department of Chemistry, Technische Universität Darmstadt, Darmstadt, Germany.
Heidrich
Nicolas G
NG
Dencher
Norbert A
NA
Seelert
Holger
H
D000251:Adenosine Triphosphatases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007854:Lead / Q000032:analysis
D007854:Lead; C036682:lead phosphate; D000251:Adenosine Triphosphatases