ATP 检测方法

Determination of ATP and ADP Levels in cells

Stock solutions:

1. Stock solutions for ATP masurements:

1.Lysing buffer: 100 mM Tris, 4 mM EDTA, pH7.75

200 ml dH2O + 3.04 g Tris + 0.304 g EDTA, pH7.75

2. Luciferase reagent: Carefully add 10 ml dH2O into bottel 1. Incubate for 5 min at 0 o C

for 5 min. Mix by very gentlely rotate the bottel (no shaking). Aliquot the reagents and stored at –20 o C for one week.

3. ATP stock: 1.04 ml dH2O is added into bottel 2 containing 10.43 mg ATP. Final concentration: 10 mg/ml or 16.5 mM. Aliquots to 8 tubes, stored in –20 oC (stable for at least 4 weeks).

4. ATP standards (put at –20 oC, stable for 3 days):

1)100 uM: 10 ml Tris buffer + 60.6 ul 16.5 mM ATP

2)10 uM: 9 ml buffer + 1 ml 100 uM ATP

3) 2.5 uM: 9.75 ml buffer + 0.25 ml 100 uM ATP

4) 1 uM: 9 ml buffer + 1 ml 10 uM ATP

5)0.5 uM: 9.5 ml buffer + 0.5 ml 10 uM ATP

6)250 nM: 9.75 ml buffer + 0.25 ml 10 uM ATP

7)100 nM: 9 ml buffer + 1 ml 1 uM ATP

8)50 nM: 9.5 ml buffer + 0.5 ml 1 uM ATP

9)10 nM: 9 ml buffer + 1 ml 100 nM ATP

10) 0 nM: buffer only

Before experiment, luciferase stock is diluted 10 fold in ddH2O.

Experimental procedures:

1.After removal of media, cells are lysed by adding 1 ml of boiling Tris buffer to each

well. Scratch until cell are lysed. Then transfer the samples to eppendoff tubes,

centrifuge at 14,000 g for 5 min.

2. Put the samples in scintillation counter. Set up the counter at: 10 sec reading, SPM.

3. Protein determination: Lyse three sister wells of each condition in 0.1N NaOH/0.1% SDS. Determine protein concentrations using BCA assay. Average the protein concentration of three samples from each condition.