Proteome Profiler Human Phospho-RTK Array Kit Best Seller

A membrane-based antibody array for the parallel determination of the relative phosphorylation of human receptor tyrosine kinases. Validated for analyte detection in cell lysates.

Troubleshooting Guide

Principle of the Assay

The Proteome Profiler Human Phospho-RTK Array Kit is a membrane-based sandwich immunoassay. Capture antibodies spotted in duplicate on nitrocellulose membranes bind to specific target proteins present in the sample (Step 1). Tyrosine phosphorylation of the captured proteins is detected with an HRP-conjugated pan phospho-tyrosine antibody (Step 2) and then visualized using chemiluminescent detection reagents (Step 3). The signal produced is proportional to the amount phosphorylation in the bound analyte.

Why Use an Antibody Array to Detect Receptor Phosphorylation?

Determining the phosphorylation of multiple receptors in a single sample can be expensive, time consuming and can require specialized equipment. Performing multiple immunoprecipitations and Western blots requires time, labor, and reagents. The use of a multiplex antibody array to detect multiple phosphorylations in a single sample can be cost-effective and also save time and sample.

• 4 Array Membranes
• 4-Well Multi-dish
• Array Buffers
• Lysis Buffer
• Wash Buffer
• Anti-Phospho-Tyrosine-HRP Detection Antibody
• Chemiluminescent Detection Reagents
• Transparency Overlay Template
• Detailed Protocol

For a complete list of the kit contents and necessary materials, please see the Materials Provided/Other Supplies Required sections of the product datasheet.

Stability and Storage

Store the unopened kit at 2 ^°C to 8 ^°C. Do not use past kit expiration date.

Assays for Analytes represented in the Proteome Profiler Human Phospho-RTK Array Kit

Scientific Data Examples for Proteome Profiler Human Phospho-RTK Array Kit

Detection of Human Receptor Tyrosine Kinase Phosphorylation in KATO-III Cell Line. KATO-III human gastric carcinoma cells were either untreated or treated with 100 ng/mL rhFGF acidic and 1 μg/mL heparin for 15 minutes. Cells for rhFGF acidic/Heparin treatment either received pre-treatment with the reversible FGF R and VEGF R inhibitor PD 173074 (Tocris, Catalog # 3044), or with the FGF R-specific tyrosine kinase inhibitor SU 5402 (Tocris, Catalog # 3300), or no pre-treatment.

The Human Phospho-RTK Array is specific for ErbB2, ErbB3, and ErbB4 as shown by receptor competition. MDA-MB-453 human breast cancer cells were treated with 100 ng/mL of rhNRG1-beta1/HRG1-beta1 (R&D Systems, Catalog # 396-HB) for 5 minutes. 5 µg of rhErbB2 (R&D Systems, Catalog # 1129-ER), rhErbB3 (R&D Systems, Catalog # 348-RB), or rhErbB4 (R&D Systems, Catalog # 1131-ER) extracellular domains were added to 50 µg of lysate and analyzed using the Human Phospho-RTK Array. Competition of a particular ErbB receptor was observed only with the corresponding recombinant soluble receptor.