Proteome Profiler Human Phospho-Immunoreceptor Array Kit

Kit Summary

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

Troubleshooting Guide

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Principle of the Assay

The Proteome Profiler Human Phospho-Immunoreceptor 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 immunoreceptors 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.

Kit Contents

This kit contains the following azide-free, fluorochrome-conjugated antibodies to verify human stem cell pluripotency:

• 4 Array Membranes
• 4-Well Multi-dish
• Array Buffers
• Lysis Buffer
• Wash Buffer
• HRP-conjugated Anti-phospho-tyrosine 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.

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Figure 1A: The Human Phospho-Immunoreceptor Array detects multiple tyrosine phosphorylated immunoreceptors in lysates prepared from cells activated by antibodymediated cross-linking of cell-surface immunoreceptors. THP-1 human acute monocytic leukemia cells were incubated with goat anti-Fcγ RIIA antibody (R&D Systems, Catalog # AF1875) or normal goat IgG (R&D Systems, Catalog # AB-108-C) followed by incubation with a donkey anti-goat IgG antibody (R&D Systems, Catalog # AF109) for 5 minutes [Maresco, D.L. et al. (1999) J. Immunol. 162:6458].

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Figure 1B: Jurkat human acute T cell leukemia cells were incubated with a mouse monoclonal anti-CD3ε antibody (R&D Systems, Catalog # MAB100) or mouse IgG1 isotype control (R&D Systems, Catalog # MAB002) followed by incubation with a goat anti-mouse antibody (R&D Systems, Catalog # AF007) for 5 minutes. 100 µg of lysate was run on each array.