| Contributor: |
The Laboratory of Andrew Murray at Harvard University
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| This protocol is intended to monitor ATP in cell extracts (i.e. to measure ATP levels in an ATP regeneration system). |
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This protocol is intended to monitor ATP in cell extracts (i.e. to measure ATP levels in an ATP regeneration system).
1. Perform suitable assay containing approximately 1 mM ATP.
2. Pre-incubate 500 μl of GG Buffer in a microcentrifuge tube to 95°C for 5 min.
3. Add 2.5 μl of the assay to the 500 μl of GG buffer and incubate at 95°C for 3 min (see Hint #1).
4. Set up luminometer for measurement:
Use default detection settings,
Clean injector thoroughly:
Run machine five times with a solution sample of 20 X SSC,
Followed by five runs with distilled, deionized water (ddH2O).
Test cleanliness of system by running a small amount of luciferin in ddH2O and examining the injected solution. The color of the post-injection solution should be similar to the pre-injection solution. If color changes to a greenish color, repeat cleaning steps.
5. Set up and measure a standard curve of ATP Solution with the prepared luminometer:
Add to separate microcentrifuge tubes:
0 μl of ATP Solution
5 μl of ATP Solution
10 μl of ATP Solution
20 μl of ATP Solution
40 μl of ATP Solution
60 μl of ATP Solution,
Bring up the final volume of all samples to 200 μl with GG Buffer.
Measure each sample twice in the luminometer (see Hint #2).
6. Set up serial dilutions of the experimental sample from Step (3) and measure them in the luminometer. Calculate concentration of ATP based on comparison with standard curve of Step (5).
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| 20 X SSC |
| pH 7.2
3 M NaCl
0.3 M Sodium Citrate
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| Luciferin |
| 20 mM MES, pH 6.5
4 mg/ml FLE-50
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| ATP Solution: |
| 0.5 uM ATP in GG Buffer.
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| GG Buffer: |
| 15 mM MgCl2
75 mM Glycylglycine, pH 8.0
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FLE-50
ATP
Glycylglycine
Magnesium Chloride
Sodium Chloride
Sodium Citrate
MES
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1. This step rapidly quenches any ATPases that might be present in samples. ATP is stable under these conditions.
2. In general, measurement in the luminometer should be linear up to 1 x 106 Relative Light Units (RLUs).
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