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 substrateStop solution
Reagent Preparation
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.
Assay Procedure
Seed 10,000-30,000 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 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.
Target/Description
Receptor tyrosine-protein kinase erbB-3, also known as HER3 (human epidermal growth factor receptor 3), is a membrane bound protein that in humans is encoded by the ERBB3 gene.
Restrictions
For research use only. Intended for use by laboratory professionals.
Datasheet