Introduction
The RayBio® Oxidated GAPDH ELISA Kit uses the "biotin probes" method to biotin label carbonyl groups of oxidized proteins. In this method, the biotin probes first react with carbonyl groups of oxidized proteins to form unstable Schiff bases, which are then further reduced to more stable amines. The biotinylated proteins can then be detected by ELISA. An anti-pan GAPDH antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells, and GAPDH present in a sample is bound to the wells by the immobilized antibody. The wells are washed, and HRP-conjugated streptavidin is used to detect oxidated GAPDH. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of oxidated GAPDH 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-GAPDH Antibody | 1 plate |
| Positive Control | 1 vial |
| HRP-Conjugated Streptavidin | 200 ÎĽL |
| Wash Buffer | 25 mL |
| Assay Diluent | 15 mL |
| Lysis Buffer | 5 mL |
| TMB One-Step Substrate Reagent | 12 mL |
| Stop Solution | 8 mL |
| Oxidation Buffer A | 12 mL |
| Oxidation Buffer B | 3 mL |
| Oxidation Labeling Reagent | 1 vials |
| Oxidation Stabilizing Reagent | 1 vials |
Additional Materials Required
- 20% (v/v) trichloroacetic acid (TCA) solution, ice-cold
- 1:1 (v/v) ethanol:ethyl acetate
- Benchtop centrifuge and microcentrifuge (4°C)
- Microplate reader capable of measuring absorbance at 450 nm
- Protease and Phosphatase inhibitors
- Precision pipettes to deliver 2 ÎĽL 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 positive control or sample dilutions
- Distilled or deionized water
- Shaker
Assay Procedure Summary
- Prepare all reagents, samples and Positive Control as instructed.
- Label samples with Oxidation Labeling Reagent
- Add Oxidation Stabilizing Reagent to samples.
- Precipitate proteins with ice-cold 20% TCA.
- Wash protein pellet twice with 1:1 (v/v) ethanol:ethyl acetate.
- Resuspend protein pellets in Oxidation Buffer B.
- Add 100 μL sample and Positive Control to each well. Incubate 2.5 hours at room temperature or overnight at 4°C with gentle shaking.
- Add 100 ÎĽL prepared HRP-Conjugated Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
- Add 100 ÎĽL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
- Add 50 ÎĽL 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.
