AffiELISA® Inorganic Pyrophosphatase ELISA Kit
AffiELISA® Inorganic Pyrophosphatase ELISA Kit provides a sensitive and specific method for the quantification of inorganic pyrophosphatase (IPPase) activity in various biological samples.
The kit is designed to deliver reliable, reproducible results, making it an ideal tool for research, clinical studies, and quality control applications in biotechnology and pharmaceutical industries. By offering high specificity, the Inorganic Pyrophosphatase ELISA Kit ensures accurate analysis of IPPase activity, contributing to better understanding and optimization of metabolic processes and enzyme function.
Intended Use:
AffiELISA® Inorganic Pyrophosphatase ELISA Kit is designed for the quantitative measurement of inorganic pyrophosphatase (IPPase) activity in biological samples.
Kit components:
| Component | Description |
| Microplate (96 wells) | Pre-coated with capture antibody specific to inorganic pyrophosphatase (IPPase) for enzyme immobilization. |
| Standards | Known concentrations of inorganic pyrophosphatase for generating a standard curve. |
| Samples | Biological samples (serum, plasma, cell lysates, etc.) containing inorganic pyrophosphatase to be measured. |
| Detection Antibody | Biotinylated antibody that binds specifically to inorganic pyrophosphatase for detection. |
| HRP-Streptavidin | HRP-labeled streptavidin that binds to the biotinylated detection antibody, enabling colorimetric detection. |
| Wash Buffer | Solution used to wash the wells, removing unbound substances between incubation steps. |
| Substrate Solution (TMB) | Tetramethylbenzidine (TMB) substrate that reacts with HRP, producing a color change for detection. |
| Stop Solution (Acidic) | Acidic solution used to terminate the reaction, changing the color from blue to yellow. |
Materials Required But Not Supplied:
- Microplate reader capable of reading absorbance at D450/650 nm.
- Microplate shaker.
Procedure workflow:
1. Prepare Reagents
- Prepare all reagents according to the kit instructions, including wash buffer, detection antibody, HRP-streptavidin, standards, and samples.
2. Add Standards and Samples
- Add 100 µL of prepared standards and samples into the appropriate wells of the microplate.
3. First Incubation
- Incubate the plate at room temperature (25°C) with shaking at 500 rpm for 60 minutes.
4. First Wash
- Discard the liquid, blot the plate dry, add 300 µL of wash buffer per well, let stand 15–30 seconds, discard, and blot dry. Repeat this wash step 3 times in total.
5. Add Detection Antibody
- Add 100 µL of the 1X detection antibody solution to each well.
6. Second Incubation
- Incubate at room temperature (25°C) with shaking at 500 rpm for 60 minutes.
7. Add HRP-Streptavidin
- Add 100 µL of the 1X HRP-streptavidin solution to each well.
8. Third Incubation
- Incubate the plate at room temperature with shaking at 500 rpm for 30 minutes.
9. Second Wash
- Perform the same washing procedure as in step 4, repeating it 3 times.
10. Add Substrate Solution
- Add 100 µL of TMB substrate solution to each well.
11. Substrate Incubation
- Incubate the plate at 37°C in the dark for 10 minutes.
12. Add Stop Solution
- Add 50 µL of stop solution to each well to terminate the enzymatic reaction.
13. Read Absorbance
- Measure absorbance at 450/650 nm using a microplate reader for data analysis.