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**ATP Enzyme Test Kit Instructions**
This ATP enzyme test kit is designed to measure the activity of various ATPases, including Naâº/Kâº-ATPase, Ca²âº-ATPase, Mg²âº-ATPase, and Ca²âº/Mg²âº-ATPase. It allows for the analysis of ATPase activity in erythrocyte membranes and tissue homogenates without the need for high-speed centrifugation. This method is ideal for laboratories seeking a simplified and efficient approach to ATPase measurement.
**Equipment Required:**
- Test tubes
- Micropipettes
- Vortex mixer
- 37°C constant temperature water bath
- Visible spectrophotometer (660 nm)
**Application Scope:**
The kit can be used to measure ATPase activity in both erythrocyte membranes and tissue homogenates, eliminating the need for complex sample preparation techniques.
**Importance of Measurement:**
ATPases are integral membrane proteins that play a crucial role in cellular functions such as ion transport, energy conversion, and signal transduction. Measuring their activity provides valuable insights into cellular metabolism and membrane integrity.
**Simplified Procedure:**
1. **Enzymatic Reaction:**
Prepare the following tubes with the specified volumes:
| Tube | A Liquid (μL) | B Liquid (μL) | C Liquid (μL) | D Liquid (μL) | E Liquid (μL) | Sample (μL) | Reagent VII (μL) |
|------|---------------|---------------|---------------|---------------|---------------|-------------|------------------|
| A | 250 | - | - | - | - | 200 | 50 |
| B | - | 250 | - | - | - | 200 | 50 |
| C | - | - | 250 | - | - | 200 | 50 |
| D | - | - | - | 250 | - | 200 | 50 |
| E | - | - | - | - | 250 | 200 | 50 |
Mix all tubes thoroughly and incubate at 37°C for 10 minutes.
2. **Phosphorus Assay:**
After incubation, mix the samples again, centrifuge at 3000–4000 rpm for 10 minutes, and take 200 μL of the supernatant for further analysis.
Prepare standard tubes as follows:
| Tube | Phosphorus Standard (0.5 μmol/mL) (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:**
- Tube A serves as the control.
- Tube B measures Naâº/Kâº-ATPase activity.
- Tube C measures Mg²âº-ATPase activity.
- Tube D measures Ca²âº-ATPase activity.
- Tube E measures combined Ca²âº/Mg²âº-ATPase activity.
Due to ongoing debates about whether Ca²âº-ATPase and Mg²âº-ATPase can be separated, if no separation is required, Tube E can represent both activities. If separation is needed, Tubes C and D can be used individually to measure each enzyme’s activity.
**Calculation Formula:**
For detailed calculation instructions, please visit our website or contact our sales team for personalized guidance.
This kit is guaranteed to provide 100 reactions for each of the A, B, C, D, and E tubes. The procedure has been optimized for accuracy, speed, and ease of use in laboratory settings.