RayBio^® MAPK Pathway ELISA Sample Kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human, mouse and rat cell lysates. By determining phosphorylated ERK1/2, JNK and P38α protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.

This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human, mouse and rat phospho-ERK1/2, total ERK1/2, phospho-JNK, total JNK, phospho- P38α and total P38α. For each target, a capture antibody has been coated onto microwells. Samples are pipetted into the wells and target protein present in a sample is bound to the wells by the immobilized antibody. The wells are washed and a detection antibody is used to detect the captured target protein. After washing away unbound antibody, an HRP-conjugated secondary antibody 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 target protein bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

• Simultaneously measure Phosphorylated protein and pan protein in one experiment (for normalization purpose)
• Screen numerous different cell lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

Phosphorylated: ERK1/2 (T202/Y204), JNK (T183/Y185) and P38α (T180/Y182)
Total: ERK1/2, JNK and P38α

• Phospho-ERK/JNK/P38α Microplate
• Positive Control
• Anti-phospho-ERK1/2 Detection Antibody
• Anti-pan ERK1/2 Detection Antibody
• Anti-phospho-JNK Detection Antibody
• Biotinylated anti-JNK Detection Antibody
• Anti-phospho-p38α Detection Antibody
• Biotinylated Anti-p38α Detection Antibody
• HRP-Conjugated anti-rabbit IgG
• HRP-Conjugated Streptavidin
• Wash Buffer
• Assay Diluent
• Lysis Buffer
• TMB One-Step Substrate Reagent
• Stop Solution

• 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 standard or sample dilutions
• Distilled or deionized water
• Shaker

1. Prepare all reagents and samples as instructed in the manual.
2. Add 100 μL of each positive control and sample to each well.
3. Incubate 2.5 hours at room temperature or overnight at 4ºC with gentle shaking.
4. Add 100 μL prepared detection antibody to each well.
5. Incubate for 1 hour at room temperature with gentle shaking.
6. Add 100 μL prepared HRP-conjugated anti-rabbit or streptavidin solution.
7. Incubate for 1 hour at room temperature with gentle shaking.
8. Add 100 μL TMB One-Step Substrate Reagent to each well.
9. Incubate 30 minutes at room temperature.
10. Add 50 μL Stop Solution to each well.
11. Read at 450 nm immediately.

The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.