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Mot1 ATPase assay Each reaction contains

Summary: Auble Lab
Mot1 ATPase assay
Each reaction contains:
4 ul 5 X BB
1 ul 20 mM DTT
1 ul 500 uM cold ATP
0.2 ul gamma 32P ATP (NEN NEG 502A, 10 mCi/ml, 3000 Ci/mmol)
2.0 ul 1% Brij 58
0.5 ul 0.5 ug/ul poly dG-dC
Use pencil to lightly mark Bakerflex PEI TLC plates with a line 2.5 cm from
bottom edge and make light marks for origins at 2 cm intervals along the line (9
spots per plate). Set up one tube for each time point which contains 0.5 ul 20
mM EDTA and 0.5 ul 1 mM cold ATP. Remove 2 ul reaction for zero time point,
mix with EDTA/ATP, quick spin and spot 2 ul on TLC plate.
Initiate reactions by addition of Mot1 to 7 nM final concentration +/- TBP (Km
for TBP is 50-100 nM)
For total [ATP] in the range of 1- 50 uM, take 8 time points at 1.25 minute
intervals (10 minute time course). For [ATP] greater than 50 uM, take time
points at 2 minute intervals (16 minute time course). Develop TLCs in 0.6 M
KH2PO4 (pH 3.4). (81.65 g/l; pH with HCl) Running solvent to about half


Source: Auble, David - Department of Biochemistry and Molecular Genetics, University of Virginia


Collections: Biology and Medicine