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2 These enzymes are shipped individually from the manufacturer as a suspension in an ammonium sulfate solution. Before taking an aliquot, the vials containing these enzymes must be agitated to resuspend the enzymes.
3 The ADP solution is prepared just before use and kept on ice. We use a fresh, unopened package of ADP for each premix preparation. The ADP can partially decompose with time to give adenosine monophosphate (AMP) and inorganic phosphate. The inorganic phosphate can be used by the enzyme premix to make nonradiolabeled ATP, which can substantially lower the specific activity of the synthesized [gamma-32P]ATP.
4 RNAse Solution. RNAse A must be heat-treated to deactivate any DNAses present. Dissolve RNAse A powder in ddH2O to a concentration of 10 mg/mL, heat this solution in a water bath for 10 minutes at 90°C, and then cool slowly to room temperature. Sufficient RNAse T1 is then added to the RNAse A solution to yield a final RNAse T1 concentration of 10 units/mL. Aliquot and freeze the solution at -80°C. The solution is stable for at least 3 months.
5 To prepare the water-saturated n-butanol, place equal volumes of ddH2O and n-butanol in a container and vortex vigorously. The top layer will be water-saturated n-butanol and the lower layer will be n-butanol-saturated water.
6 About 50 µCi of [gamma-32P]ATP will be sufficient radioactivity for labeling approximately 30-50 samples.
7 The nonradioactive ATP solution is prepared by dissolving 10 mg of fresh ATP in 20 mL of ddH2O to give a concentration of 0.907 nmol ATP/µL water. It is important that the nonradioactive ATP used to make this solution has not decomposed, otherwise, the spec. act. will be low. Also, the amount of radioactive [gamma-32P]ATP is negligible compared to the amount of nonradioactive ATP used and will not influence the calculation of ATP concentration.
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