Nucleotide Binding/Hydrolysis Assay
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| Nucleotide mix |
| Motor (50 - 100 µM; purity > 95%) |
| 0.5 M Tris-OAc, pH 7.5 |
| 10 mM EGTA |
| 10 mM MgCl2 |
| DDW |
| Sephadex G-50 Medium column (0.8 cm in x 20 cm) |
| Column buffer = |
50 mM Tris-OAc, pH 7.5 1 mM EGTA 1 mM MgCl2 |
| Scintillation fluid |
| Scintillation vials and disposable inserts |
| 1. | Prepare reaction mixes (mutant and wild-type) by mixing the following: |
|
__ µL 1 µL 10 µL 10 µL 10 µL __ µL ______ 100 µL |
50 - 100 µM motor gamma or alpha-32 PATP nucleotide mix 0.5 M Tris-OAc, pH 7.5 10 mM EGTA 10 mM MgCl2 DDW |
|
Adjust the volume of the motor to give 5 - 10 µM in the mix and adjust the volume of DDW to give the final volume of 100 µL. Centrifuge the motor before adding to the mix. Incubate the mix on ice overnight. Prepare reaction mixes for both mutant and wild-type motors on the same day. |
| 2. | Apply reaction mix to a Sephadex G-50 Medium column. |
| 3. | Collect two 1 mL fractions and label them fr 1 and fr 2, then collect 28 x 5-drop fractions (each = ~250 µL) and label them fr 3 - fr 30. |
| 4. | Take 50 µL of fractions 3 - 22 and mix with 0.5 mL of scintillation fluid in a disposable insert. Put insert into a scintillation vial, precounted to determine background of less than 30 cpm, then count the vial on the 32 P channel. |
| 5. | Determine protein concentration of each fraction by adding 50 µL of concentrated Bio-Rad reagent to the remainder of fractions 3 - 12 (~200 µL). Prepare a blank by adding 50 µL of concentrated Bio-Rad reagent to 200 µL of column buffer. Read each fraction in a microcuvette at OD595 . Cuvette can be rinsed out with DDW between each reading. |
| 6. | Plot fraction number versus cpm and OD595 . |
| 1. | For Km determination, the concentration (µM) of bound and unbound alpha-32 PATP can be determined from the starting ATP concentration. |
| 2. | Co-elution of cpm with the protein peak is interpreted as bound ADP, and unbound cpm is ATP + ADP + Pi . |
Modified from Song and Endow 1998 Nature 396, 587-590.
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Materials
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Procedure
Notes
