Z'-LYTE Kinase Assay Kits

Protein kinases and phosphatases regulate many critical biological mechanisms, including cell growth, proliferation, differentiation and metabolism, by protein phosphorylation and dephosphorylation. Aberrations in the activity of the kinases and phosphatases involved in signal transduction have been linked to many human diseases. The discovery of more than 600 kinases and phosphatases encoded by the human genome has spurred development of rapid screening techniques for potential drugs against these enzymes.

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To address this need, the Z´-LYTE technology has been developed. This robust homogeneous assay method is based on Fluorescence Resonance Energy Transfer (FRET) and requires no expensive radioactive or antibody-based reagents. The proprietary Z´-LYTE technology is highly compatible with automated high-throughput screening systems. Invitrogen offers numerous Z´-LYTE FRET-peptide substrates in convenient platform-ready assay formats. New FRET-peptide substrates, and their associated Z´-LYTE assays, can be designed and developed quickly when the amino acid sequence of the substrate is known. Therefore, the Z´-LYTE technology can readily meet the growing demand for new assays to screen for inhibitors of a broad array of tyrosine and serine/threonine protein kinases.

• Signal transduction and drug discovery research
• High-throughput screening
• Quantitative measurement of inhibitor IC50 values
• Screening of novel tyrosine and serine/threonine kinase inhibitors
• Selectivity profiling
• Custom peptide development

• Broad kinase coverage
• Rapid assay development
• Robust and reproducible results – Z´factor values >0.7
• Conserve reagents and money through miniaturization

Figure 1. Z'-LYTE Illustration

The Z´-LYTE biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage (Figure 1).

In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET-peptides. Phosphorylation of FRET-peptides suppresses cleavage by the Development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET. A ratiometric method, which calculates the ratio (the Emission Ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is used to quantitate reaction progress, as shown in the equation below.

A significant benefit of this ratiometric method for quantitating reaction progress is the elimination of well-to-well variations in FRET-peptide concentration and signal intensities. As a result, the assay yields very high Z´-factor values (>0.7) at a low percent phosphorylation.

Both cleaved and uncleaved FRET-peptides contribute to the fluorescence signals and therefore to the Emission Ratio. The extent of phosphorylation of the FRET-peptide can be calculated from the Emission Ratio. The Emission Ratio will remain low if the FRET-peptide is phosphorylated (i.e., no kinase inhibition) and will be high if the FRET-peptide is non-phosphorylated (i.e., kinase inhibition).

The Z´-LYTE Detection Kinase Assay Kits correspond to the specific Z´-LYTE Ser/Thr and Tyrosine Peptides identified in the Z’-LYTE Substrate Panels. Once the optimal peptide has been selected from the Panel, the corresponding kit may be ordered to conduct screens for inhibitors of kinase activity. These kits may also be used for selectivity profiling of kinase inhibitors. The Reactivity Table also gives guidance as to which peptide is appropriate to assay the activity of a particular Ser/Thr or Tyr kinase.

To see a summary protocol of each kinase we have validated in the Z'-LYTE system, view the Customer Protocol and Assay Conditions document. To view the customer protocol for each individual kit, please click on the catalog number for the kit of interest, then click on the name of the kit in the table that appears.

If no Z´-LYTE Peptide Substrate worked with a desired kinase protein, but the sequence of its substrate is known, a custom Z´-LYTE Peptide may be synthesized and validated.

Custom quantities of peptide substrates and kit components are available for larger screening requirements at special discount pricing.

*Z'-LYTE is covered by one or more of the following U.S. patents, as well as corresponding pending or issued foreign patents: 6,410,255; other U.S. patents pending.

Tyrosine Kinase Assays

Serine/Threonine Kinase Assays