Introduction
The RayBio® SUMOylated-Peroxiredoxin 6 and Total-Peroxiredoxin 6 ELISA Kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human sumoylated ATP-citrate synthase and total ATP-citrate synthase. An anti-pan Peroxiredoxin 6 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and Peroxiredoxin 6 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-sumoylation antibody is used to detect sumoylated Peroxiredoxin 6 or biotinylated anti-Peroxiredoxin 6 antibody is used to detect total Peroxiredoxin 6 . After washing away unbound antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of sumoylated Peroxiredoxin 6 or total Peroxiredoxin 6 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Product Features
- Quantify quickly and easily
- Screen numerous different lysates without performing a Western Blot analysis
- Minimal hands-on time, convenient, and non-radioactive material
Kit Components
| Component | Size / Qty |
|---|
| Microplate Coated With Anti-Pan-Peroxiredoxin 6 Antibody | 1 plate |
| Anti-SUMOylation Detection Antibody | 1 vial |
| Anti-Peroxiredoxin 6 Detection Antibody | 1 vial |
| HRP-Conjugated Streptavidin | 200 µL |
| Wash Buffer | 25 mL |
| Assay Diluent | 15 mL |
| Lysis Buffer | 5 mL |
| TMB One-Step Substrate Reagent | 12 mL |
| Stop Solution | 8 mL |
Additional Materials Required
- Microplate reader capable of measuring absorbance at 450 nm
- Protease and Phosphatase inhibitors
- Precision pipettes to deliver 2 µL to 1 mL volumes
- Adjustable 1-25 mL pipettes for reagent preparation
- 100 mL and 1 liter graduated cylinders
- Log-log graph paper or computer and software for ELISA data analysis
- Absorbent paper
- Tubes to prepare sample dilutions
- Distilled or deionized water
- Shaker
Assay Procedure Summary
- Prepare all reagents and samples as instructed.
- Add 100 µL sample to each well. Incubate 2.5 hours at room temperature or overnight at 4°C with gentle shaking.
- Add 100 µL prepared detection antibody to each well. Incubate for 1 hour at room temperature with gentle shaking.
- Add 100 µL prepared HRP-Conjugated Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
- Add 100 µL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
- Add 50 µL Stop Solution to each well. Read at 450 nm immediately.
Storage/Stability
The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.
Customization
This assay can be developed for other species (mouse, rat, etc.) upon request. Contact
techsupport@raybiotech.com for more information.