Introduction
Protein phosphorylation is instrumental in the regulation of protein activity within a cell. It plays important roles in the living cells including proliferation, differentiation and metabolism. A large number of protein kinases and phosphatases have been extensively investigated, and have been shown to be involved in signal transduction pathways.
The RayBio® Cell-Based Human/Mouse MKK6 (S207) ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cells. It can be used for measuring the relative amount of MKK6 (S207) phosphorylation and screening the effects of various treatments, inhibitors (such as siRNA or chemicals), or activators in cultured human, mouse cell lines. By determining MKK6 protein phosphorylation in your experimental model system, you can verify pathway activation in your cell lines without spending excess time and effort in preparing cell lysate and performing an analysis of Western Blot. In the Cell-Based MKK6 (S207) ELISA kit, cells are seeded into a 96 well tissue culture plate. The cells are fixed after various treatments, inhibitors or activators. After blocking, Anti-Phospho-MKK6 (S207) or Anti-MKK6 is pipetted into the wells and incubated. The wells are washed, and HRP-Conjugated anti-rabbit IgG and HRP-Conjugated anti-mouse IgG are added to the corresponding wells. The wells are washed again, a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Product Features
- Site and signal pathway-specific
- In vitro detection of adherent cell culture
- No sample lysis needed
- Compatible with a standard ELISA plate reader
- Faster results than with ELISA
- Adaptable for high-throughput screening and drug discovery
Application Notes
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
- Seed 10,000-30,000 cells into each well and incubate overnight.
- Apply various treatment, inhibitors or activators according to manufacture's instructions.
- Add 100 µl of Fixing Solution into each well and incubate for 20 min at RT with shaking.
- Add 200 µl of prepared 1X Quenching Buffer and incubate 20 min at RT.
- Add 200 µl of Blocking Solution and incubate for 1 h at 37 °C.
- 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.
- Add 50 µl of prepared 1X HRP-Anti-Rabbit or Mouse IgG and incubate for 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Storage/Stability
-20°C