Experimental instruments
Test tube, Micropipettor, Vortex mixer, 37℃ water bath/gas bath, Spectrophotometer (636 nm)
Application
This kit can be used for detection of Ca2+-ATPase activity in animal erythrocyte, whole blood and other samples.
Detection significance
ATPase exists on the membrane of tissue cells and organelles. It is a kind of protease on the biological membrane which plays an important role in material transport, energy conversion and information transmission. The enzyme activity of ATPase will have a series of changes when the body in the hypoxic or diseases condition, and is also associated with some genetic diseases.
Operation steps
1. Enzymatic reactions
| Control tube | Ca2+-ATPase detection tube |
Reagent 1 (μL) | 70 | 70 |
Reagent 2 (μL) | 20 |
|
Reagent 3 (μL) |
| 20 |
Reagent 4 (μL) | 20 | 20 |
Reagent 5 (μL) |
| 20 |
Reagent 6 (μL) | 20 |
|
Sample (μL) |
| 100 |
Mix, react for exactly 10 min at 37℃. | ||
Reagent 7 (μL) | 50 | 50 |
Sample (μL) | 100 |
|
Mix, centrifuge for 10 min at 3500 rpm and take the supernatant to determine phosphorus.
2. Determine phosphorus
| Blank tube | Standard tube | Control tube | Ca2+-ATPase detection tube |
Double-distilled water (mL) | 0.15 |
|
|
|
0.02 μmol/mL phosphorus standard solution (mL) |
| 0.15 |
|
|
Supernatant (mL) |
|
| 0.15 | 0.15 |
Phosphorus determination application solution (mL) | 0.5 | 0.5 | 0.5 | 0.5 |
Mix and stand for 2 min at room temperature. | ||||
Stop solution (mL) | 0.5 | 0.5 | 0.5 | 0.5 |
Mix and stand for 5 min at room temperature. Measure the OD values of each tube at 636 nm wavelength with 0.5 cm diameter cuvette, set to zero with double-distilled water.
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