Determination of a Marker Enzyme Ouabain-Sensitive Na,K-ATPase

1 Determination of a Marker Enzyme Ouabain-Sensitive Na,K-ATPase 

7 mM NaCl, 26.7 mM KCl, 6.3 mM NaNj, 4 mM MgCh, 0.13 mMEGTA,40 mM imidazole. Dissolve required amounts of the solids in about 8 ml ddH20, adjust pH 7.2 and fill up with ddH20 to 10 ml 

5% ammonium molybdate (w/v) in ddH20 10% ascorbic acid (w/v) in ddH2 0 

Dilute sarcolemmal vesicles from Protocol 5.3.2 to about 1 mg pro-tein/ml with Soln. A (for protein concentration determination the Lowry method is recommended). Add 1 pi of Soln. B per 100 pi SL dilution, vortex and put on ice. Pipet the probes in triplicates into Eppendorf tubes according to Table 5.6. Label the ouabain-containing tubes with those without ouabain should be signed 

Incubate the probes at 37 °C for 5 min, then start the enzymatic reaction by addition of 15 pi of SL dilution to each -I- and Shake the probes at 37 °C and stop by addition of 0.5 ml Soln. D after exactly 15 min. 

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