RayBio® Cell-Based Human/Mouse/Rat Stat (1, 3, and 5) Phosphorylation ELISA Kit 2 x 96-Well Kit

Kit Components

• Cell Culture Microplate
• Wash Buffer A
• Wash Buffer B
• Fixing Solution
• HRP-Conjugated Secondary Antibody
• Blocking Buffer
• TMB One-Step Substrate
• Stop Solution
• Quenching Buffer

Other Materials Required

• Distilled or deionized water
• 100 ml and 1 liter graduated cylinders
• Tubes to prepare sample dilutions
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Adjustable 1-25 ml pipettes for reagent preparation
• Benchtop rocker or shaker
• Microplate reader capable of measuring absorbance at 450 nm

Protocol Outline

1. Seed 10,000-30,000 cells into each well and incubate overnight.
2. Apply various treatment, inhibitors or activators according to manufacture’s instructions.
3. Add 100 µl of Fixing Solution into each well and incubate for 20 min at RT with shaking.
4. Add 200 µl of prepared 1X Quenching Buffer and incubate 20 min at RT.
5. Add 200 µl of Blocking Solution and incubate for 1 h at 37 °C.
6. Add 50 µl of 1X anti-phospho-protein specific antibody or anti-pan-protein specific antibody to each well and incubate for 2 h at RT.
7. Add 50 µl of prepared 1X HRP-Anti-Rabbit or Mouse IgG and incubate for 1 h at RT.
8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 µl of Stop Solution to each well.
11. Read at 450 nm immediately.