ATPase Assays with 32P-ATP
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Materials
| Purified Motor Protein, 20-80 µM |
| Nucleotide Mix = |
50 mM Mg・ATP gamma-32 P-ATP to give 5 000 - 10 000 cpm/nmol 10 mM HEPES, pH 7.2 1 mM EGTA 1 mM MgCl2 |
| 5% Norit A activated charcoal in 50 mM NaH2 PO4 |
| Reaction Mix = |
___µl motor = 0.5 - 1 µM final concentration (__µl microtubules) 10 µl nucleotide mix = 5 mM Mg・ATP final concentration 30 µl of 100 mM HEPES, pH 7.2 = 10 mM HEPES, pH 7.2 30 µl of 10 mM EGTA = 1 mM EGTA 30 µl of 10 mM MgCl2 = 1 mM MgCl2 30 µl of 10 mM DTT = 1 mM DTT __ µl of DDW _______ 300 µl |
Procedure
| 1. | Prepare reaction mix, add motor last, vortex briefly. |
| 2. | Take timepoints at 0, 2, 5, 10, 20, 40, 60 min by withdrawing 40 µl into microfuge tubes containing 0.76 ml of the activated charcoal suspension. |
| 3. | Vortex tubes, keep on ice 15 min, centrifuge 5 min, withdraw supernatant into new tube and centrifuge again to remove charcoal particles. |
| 4. | Count 100 µl and 200 µl samples of the supernatant to determine motor hydrolytic activity. Normalize and average the cpm/timepoint and determine the cpm for 0.8 ml. Subtract the cpm at 0 min to eliminate background. Divide by the specific activity of ATP to determine the nanomoles of Pi released at each timepoint. Divide by the nanomoles of motor protein in the 40 µl timepoint and 60 s/min to convert to kcat (s-1 ). |
| 5. | Count 2 µl and 4 µl samples of the reaction mix to determine ATP specific activity. Specific activity = cpm/µl ?5 nm ATP/µl. |
| 6. | Plot kcat (y axis) versus time (x axis) and curve fit with the Michaelis-Menton equation to determine Vmax . |
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