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ATP Enzyme Test Kit Instructions
(Measure Naâº, Kâº, Ca²âº, Mg²âº, and ATPase Activity)
**First, the Experimental Equipment:**
Test tubes, micropipettes, vortex mixer, 37°C constant temperature water bath, and a visible spectrophotometer (660 nm).
**Second, the Scope of Application:**
This kit is suitable for measuring ATPase activity in erythrocyte membranes without the need for high-speed centrifugation and in tissue homogenates that also do not require high-speed centrifugation.
**Third, the Significance of the Measurement:**
ATPase is found on cell membranes and organelle membranes. It functions as a membrane-bound enzyme involved in material transport, energy conversion, and cellular signaling, making it essential for many physiological processes.
**Fourth, Simplified Operation Steps:**
1. **Enzymatic Reaction:**
- Prepare the following tubes: A, B, C, D, and E.
- Add the reagents according to the table below:
| Tube Number | A Tube | B Tube | C Tube | D Tube | E Tube |
|-------------|--------|--------|--------|--------|--------|
| A Liquid (μl) | 250 | 250 | 250 | 250 | 250 |
| B Liquid (μl) | 250 | 250 | 250 | 250 | 250 |
| C Liquid (μl) | 250 | 250 | 250 | 250 | 250 |
| D Liquid (μl) | 250 | 250 | 250 | 250 | 250 |
| E Liquid (μl) | 250 | 250 | 250 | 250 | 250 |
| Sample (μl) | 200 | 200 | 200 | 200 | 200 |
| Reagent VII (μl) | 50 | 50 | 50 | 50 | 50 |
| Sample (μl) | 200 | 200 | 200 | 200 | 200 |
- Mix all tubes thoroughly and incubate at 37°C for 10 minutes.
2. **Phosphorus Setting:**
- After incubation, mix well, centrifuge at 3000–4000 rpm for 10 minutes, and collect 200 μL of supernatant for each tube.
**Standard Tube Preparation:**
- For the standard curve, prepare the following:
| Tube | 0.5 μmol/ml Phosphorus (μl) | Supernatant (μl) | Phosphorus (μl) |
|------|-----------------------------|------------------|-----------------|
| A | 200 | 200 | 2000 |
| B | 200 | 200 | 2000 |
| C | 200 | 200 | 2000 |
| D | 200 | 200 | 2000 |
| E | 200 | 200 | 2000 |
- Mix well, boil at 37°C for 30 minutes, cool to room temperature, and measure absorbance at 660 nm using a 1 cm path length cuvette with distilled water as the blank.
**Notes:**
- A tube is the control.
- B tube measures Naâº/Kâº-ATPase.
- C tube measures Mg²âº-ATPase.
- D tube measures Ca²âº-ATPase.
- E tube measures Ca²âº/Mg²âº-ATPase.
- The kit is designed for 100 tests per set. Due to ongoing academic debate about whether Ca²⺠and Mg²⺠ATPases can be separated, if separation is not required, E tube can represent both. If separation is needed, C and D tubes can be used individually.
**Fifth, Calculation Formula:**
For accurate results, refer to our website or contact our sales team for detailed calculation instructions and interpretation guidelines.
Please visit our official website or reach out to our customer support for further assistance and technical guidance.