EMD: Kinase Kit Resource - Kinase ELISAs and PhosphoDetect ELISAs

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Kinase Kit Resource: Kinase ELISAs and PhosphoDetect™ ELISAs

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For detecting total and phosphorylated protein kinase levels

Summary
Protein Kinase ELISAs (Enzyme-linked immunosorbent assays) measure total or phosphorylated kinase levels in biological samples, see example data in Figure 3. ELISA kits come with 96-well plates which have been coated with antibodies against the kinase to be detected (Figure 4). When the sample is added, the kinase binds to the antibody, and the plate is washed. A solution containing a detector antibody, also specific for the kinase, is added, which binds the bound kinase. For PhosphoDetect ELISAs, the detector antibody is phospho-specific, recognizing only the phosphorylated form of the protein. After an additional washing step, a secondary antibody which will recognize the bound antibody is added. This antibody is also conjugated with horseradish peroxidase (HRP), and after an additional washing step, the quantity of the protein can be detected colorimetrically by the addition of a substrate for HRP.

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When to use	Use these kits to quantitate levels of total or phosphorylated protein kinase
Advantages	• Specific, sensitive detection of kinase levels in non-purified samples • Detection can be species-specific • No special equipment required
Considerations	• Does not measure kinase activity directly • Direct inhibitors/activators cannot be used
Sample Type	Tissue homogenates, cell extracts, partially purified protein kinase samples
Detection	Colorimetric
Equipment Required	Colorimetric ELISA plate reader