Product Features
- Rapidly measure phosphorylated protein in adherent cell lines
- Simultaneously measure Phosphorylated protein and pan protein in one experiment (for normalization purpose)
- No sample lysis is needed
- Compatible with a standard ELISA plate reader
Kit Components
- Uncoated 96-well Strip Microplate
- Wash Buffers
- Fixing Solution
- Quenching Buffer
- Blocking Buffer
- Anti-phospho antibody
- Anti-pan antibody
- HRP-Conjugated Secondary Antibody
- TMB One-Step Substrate
- Stop Solution
Additional Materials Required
- A model cell line, protein tyrosine kinase inhibitors, growth factors or cytokines
- Microplate reader capable of measuring absorbance at 450 nm
- 37°C incubator
- 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
- Absorbent paper
- Distilled or deionized water
- Orbital shaker or oscillating rocker
Assay Procedure Summary
- Seed 30,000 cells into each well and incubate overnight.
- Apply various treatment, inhibitors or activators according to manufacturer's instructions.
- Add 100 µl of Fixing Solution into each well and incubate for 20 minutes at room temperature.
- Add 200 µl of prepared Quenching Buffer and incubate 20 minutes at room temperature.
- Add 200 µl of prepared Blocking Buffer and incubate for 1 hour at 37°C.
- Add 50 µl of prepared primary antibody to each well and incubate for 2 hours at room temperature.
- Add 50 µl of prepared HRP Conjugated secondary antibody and incubate for 1 hour at room temperature.
- Add 100 µl of TMB Substrate and incubate 30 minutes at room temperature.
- Add 50 µl of Stop Solution to each well. Read at 450 nm immediately.
Storage/Stability
Store entire kit at ≤ -20 °C immediately upon arrival. Kit must be used within the 6 month expiration date. Avoid repeated freeze-thaw cycles.