Nucleotide Binding/Hydrolysis Assay

互联网2013-09-05

1012

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.

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.