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Caspase-8 IETD-R110 Fluorometric HTS Assay Kit

Caspase-8 IETD-R110 Fluorometric HTS Assay Kit is specifically designed for HTS-based assays. The kit provides a homogenous assay system for fast and highly sensitive detection of caspase-8 activity by fluorescence in enzymatic reaction or mammalian cells.

Product Attributes

Apoptosis/viability marker

Caspase

For live or fixed cells

Cell lysis required

Assay type/options

Endpoint assay, Homogenous assay, High-throughput assay

Detection method/readout

Microplate reader (fluorescence)

Substrate specificity

Caspases

Colors

Green

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Product Description

Caspase-8 IETD-R110 Fluorometric HTS Assay Kit is specifically designed for HTS-based assays. The kit provides a homogenous assay system for fast and highly sensitive detection of caspase-8 activity by fluorescence in enzymatic reaction or mammalian cells. The fluorogenic substrate (Ac-IETD)2-R110 contains two IETD tetrapeptides and is completely hydrolyzed by the enzyme in two successive steps. Cleavage of the first IETD peptide results in the monopeptide Ac-IETD-R110 intermediate, which has absorption and emission wavelengths similar to those of R110 (λExEm = 496/520 nm) but has only about 10% of the fluorescence of the latter. Hydrolysis of the second IETD peptide releases the dye R110, leading to a substantial fluorescence increase.

Note: While caspase-8 preferentially cleaves the consensus sequence IETD compared to other substrate sequences, other caspases such as caspase-3 also can cleave IETD efficiently. Overlapping caspase substrate recognition limits the usefulness of caspase substrate peptides for distinguishing between different caspase activities in cell lysates.

The assay kit includes Ac-IETD-CHO, which is a caspase-8 inhibitor and can be used as a negative control. Also, R110 is provided in the kit for generating a standard curve, which can be used for quantifying caspase-8 activity.

Kit Components:

  • Cell lysis/assay buffer
  • Enzyme substrate (Ac-IETD)2-R110
  • Enzyme inhibitor Ac-IETD-CHO
  • R110

Features:

  • HTS-compatible: Single-step homogenous assay specifically designed for HTS-based detection.
  • Fast: Fast enzyme kinetics.
  • Sensitive: The enzymatic reaction forms intensely green fluorescent rhodamine 110 (R110) product.

References

1) Biochemistry 38, 13906 (1999).

 

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