Proteome Profiler Human Phospho-Kinase Array Kit

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

Troubleshooting Guide

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Principle of the Assay

The Proteome Profiler Human Phospho-Kinase 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). Phosphorylation of captured proteins is detected with biotinylated phospho-specific detection antibodies (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 kinases 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.

Each Human Phospho-Kinase Antibody Array Kit contains most of the necessary components for a ready-to-run assay without the need for specialized equipment.

• 8 Array Membranes (4 of membrane A and 4 of membrane B)
• 8-Well Multi-dish
• Lysis Buffer
• Array Buffers
• Wash Buffer
• Detection Antibody Cocktail A
• Detection Antibody Cocktail B
• Streptavidin-HRP
• Chemiluminescent Detection Reagents
• Transparency Overlay Template
• Detailed product datasheet with 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 Human Phospho-Kinase Array Kit

Figure 1. MCF-7 human breast cancer cells were either left untreated or exposed to 50 J/m2 of UV light followed by a 4 hour recovery before lysis.

Figure 2. A431 human epithelial carcinoma cells were either left untreated or treated with 200 ng/mL recombinant human (rh) EGF (Catalog # 236-EG) for 5 minutes.

Figure 3. MCF-7 human breast cancer cells were either left untreated or treated with 100 ng/mL of rhIGF-I (Catalog # 291-G1) for 1 hour.

Protein kinases are the largest class of enzymes in the human genome. These enzymes regulate almost all cellular processes by adding phosphate groups to proteins, thereby modifying the activity, localization, and overall function of their targets. Consequently, abnormal activity of kinases underlies many diseases including cancer.