Introduction
The RayBio® S-Acylated Src ELISA Kit uses a modified "biotin-switch" method to allow for the direct visualization of human S-Acylated Src by ELISA. In this S-Acylation biotin switch assay, unmodified free cysteines are first blocked. S-Acylated cysteines are then selectively reduced for specific labeling with biotin-maleimide reagents, which irreversibly bind to the cysteine thiol that was S-Acylated. Biotinylation of the newly formed thiol groups can then be detected by ELISA. An anti-pan Src antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells, and Src 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 S-Acylated Src. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of S-Acylated Src 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-Src 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 |
| S-Acylation Buffer A | 12 mL |
| S-Acylation Buffer B | 3 mL |
| S-Acylation Buffer C | 25 mL |
| S-Acylation Buffer D | 12 mL |
| S-Acylation Blocking Reagent | 2 vials |
| S-Acylation Reduction Reagent | 2 vials |
| S-Acylation Labeling Reagent | 2 vials |
Additional Materials Required
- Acetone (pre-chilled (-20°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.
- Block unmodified free cysteines using prepared S-Acylation Blocking Buffer at 50°C with gentle rocking.
- Precipitate proteins with ice-cold acetone.
- Wash protein pellet twice with cold acetone/water mixture.
- Resuspend protein pellets in Buffers and adding Reducing and Labeling Reagents.
- Precipitate proteins with ice-cold acetone.
- Resuspend protein pellets.
- 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.
