英文名 ：Amplite™ Colorimetric Total NADP and NADPH Assay Kit
供应商 ：上海研卉生物科技有限公司
库存 ：3W
规格 ：400 Tests
比色法总烟酰胺腺嘌呤二核苷酸磷酸和烟酰胺腺嘌呤二核苷酸磷酸检测试剂盒
Amplite™ 比色法总烟酰胺腺嘌呤二核苷酸磷酸和烟酰胺腺嘌呤二核苷酸磷酸检测试剂盒
英文名: Amplite™ Colorimetric Total NADP and NADPH Assay Kit
烟酰胺腺嘌呤二核苷酸（NAD+）和烟酰胺腺嘌呤二核苷酸磷酸酯（NADP+）是细胞内两种重要的辅助因子。NADH是NAD+的还原形式，NAD+是NADH的氧化形式。它通过酯键在腺苷酸的2'位置加上一个磷酸基而形成NADP。NADP用于合成代谢的生物反应，如脂肪酸和核酸的合成，需要NADPH作为还原剂在叶绿体中，NADP是光合作用的初级反应中重要的氧化剂。然后利用光合作用产生的NADPH作为光合作用Calvin循环中生物合成反应的还原力。传统的NAD/NADH和NADP/NADPH测定是通过在340nm处监测NADH或NADPH的吸收来完成的这种方法灵敏度低，干扰大，因为分析是在需要昂贵石英微板的紫外范围内进行的。该Amplite™NADP/NADPH检测试剂盒为敏感检测NADP和NADPH提供了一种方便的方法在酶循环反应中，系统中的酶特别识别NADP/NADPH。无需从样品混合物中纯化NADP/NADPH。酶循环反应显著提高了检测灵敏度。
Protocol summary
- Prepare NADP/NADPH working solution (50 µL)
- Add NADPH standards or test samples (50 µL)
- Incubate at room temperature for 15 minutes – 2 hours
- Monitor the absorbance intensity at 575 ± 5 nm
Important notes
Thaw one of each kit component at room temperature before starting the experiment.
| Instrument: |
Absorbance microplate reader |
| Absorbance: |
575 ± 5 nm |
| Recommended plate: |
Clear bottom |
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.1. NADPH standard solution (1 mM):
Add 200 µL of 1X PBS buffer into the vial of NADPH Standard (Component C) to make 1 mM (1 nmol/µL) NADPH standard solution.
NADPH standard
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/15260Add 10 µL of 1 mM (1 nmol/µL) NADPH standard solution to 990 µL 1X PBS buffer to generate 10 µM (10 pmol/µL) NADPH standard solution. Take 10 µM NADPH standard solution to perform 1:3 serial dilutions in 1X PBS buffer to get serially diluted NADPH standards (NS7 - NS1). Note: Diluted NADPH standard solution is unstable and should be used within 4 hours.
Add 10 mL of NADPH Sensor Buffer (Component B) into the bottle of NADP/NADPH Recycling Enzyme Mix (Component A) and mix well to make NADP/NADPH working solution. Note: This NADP/NADPH working solution is enough for two 96-well plates.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
Table 1. Layout of NADPH standards and test samples in a white/clear bottom 96-well microplate. NS= NADPH Standards (NS1 - NS7, 0.003 to 3 µM) , BL=Blank Control, TS=Test Samples.
| BL |
BL |
TS |
TS |
| NS1 |
NS1 |
... |
... |
| NS2 |
NS2 |
... |
... |
| NS3 |
NS3 |
|
|
| NS4 |
NS4 |
|
|
| NS5 |
NS5 |
|
|
| NS6 |
NS6 |
|
|
| NS7 |
NS7 |
|
|
Table 2. Reagent composition for each well. High concentration of NADPH (e.g., >100 µM, final concentration) may cause reduced signal due to the over oxidation of NADPH sensor.
| Well |
Volume |
Reagent |
| NS1 - NS7 |
50 µL |
Serial Dilutions (0.003 to 3 µM) |
| BL |
50 µL |
1X PBS buffer |
| TS |
50 µL |
test sample |
- Prepare NADPH standards (NS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Prepare cells or tissue samples as desired.
- Add 50 µL of NADP/NADPH working solution to each well of NADPH standard, blank control, and test samples to make the total NADPH assay volume of 100 µL/well. For a 384-well plate, add 25 µL of NADP/NADPH working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction at room temperature for 15 minutes to 2 hours, protected from light.
- Monitor the absorbance increase with an absorbance plate reader at 575 ± 5 nm or at the absorbance ratio of ~570 nm to ~605 nm to increase assay sensitivity. Note: For NADP/NADPH ratio measurements, kit 15263 is recommended. For cell based NADP/NADPH measurements, ReadiUse™ mammalian cell lysis buffer *5X* (cat #20012) is recommended to use for lysing the cells.
The reading (Absorbance (570 nm)) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate NADPH or NADH samples. We recommend using the Online Linear Regression Calculator which can be found at:
https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator
Figure 1. NADPH dose response was measured with Amplite™ Colorimetric Total NADP and NADPH Assay Kit in a white/clear bottom 96-well plate using a NOVOStar microplate reader (BMG Labtech).
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.
