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Caspase-8 IETD-R110 Fluorometric HTS Assay Kit

A fluorometric endpoint assay kit for detection of caspase-8 activity in cells by high throughput screening (HTS).

Product Attributes

Apoptosis/viability marker

Caspase

For live or fixed cells

Cell lysis required

Detection method/readout

Microplate reader (fluorescence)

Assay type/options

Endpoint assay, High-throughput assay, Homogeneous assay

Substrate specificity

Caspases

Colors

Green

Excitation/Emission

496/520 nm (end product)

Storage Conditions

Store at -10 to -35 °C

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Product Description

Caspase-3 DEVD-R110 Fluorometric HTS Assay Kit  is a homogenous assay system for fast and highly sensitive detection of caspase-3 activity in mammalian cells. The kit is specifically designed for high throughput screening (HTS)-based assays.

Features

  • Caspase-8 activity measured with substrate (Ac-IETD)2-R110
  • Designed for high throughout assays
  • Fast enzyme kinetics
  • Enzymatic reaction forms green fluorescent R110 product
  • Endpoint assay requires cell lysis

Kit Components

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

About this kit

Caspase-8 is the most upstream caspase in the CD95/Fas apoptotic pathway and is activated by the signaling pathways for CD95/Fas and TNF. The fluorogenic and chromogenic substrate (Ac-IETD)2-R110 contains two IETD tetrapeptides and is completely hydrolyzed by the caspase-8 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 (rhodamine 110) (Ex/Em= 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.

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

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.

 

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