Support & Resources

+1

Caspase-8 IETD-R110 Fluorometric and Colorimetric Assay Kit

A fluorometric and colorimetric endpoint assay kit containing a IETD substrate for detection of caspase-8 activity in cells.

Product Attributes

Apoptosis/viability marker

Caspase

For live or fixed cells

Cell lysis required

Detection method/readout

Microplate reader (absorbance), Microplate reader (fluorescence)

Assay type/options

Endpoint assay

Substrate specificity

Caspases

Colors

Green

Excitation/Emission

496/520 nm (end product)

Storage Conditions

Store at -10 to -35 °C

Size
Catalog #
price
Qty
25 assays
100 assays
Clear selection
Add to wishlist
ADD TO CART
(Please select quantity above )

Wishlist updated! View wishlist

Shipping, Handling & Tax (CA only) not included in price. Learn more

Tax is assessed in the state of California. All fees will be calculated during checkout based on your shipping information and are subject to change.

SPECIAL - Order 5 or more and receive 10% off.

Request a Bulk Quote

Protocols
SDS

Product Description

Caspase-8 Fluorometric and Colorimetric Assay Kit provides a simple assay system for fast and highly sensitive detection of caspase-8 activity either by fluorescence or absorbance in mammalian cells. Biotium also offers the Caspase- IETD-R110 Fluorometric HTS Assay Kit (30012), a one-step fluorescent assay for high throughput screening (HTS)

Features

  • Caspase-8 activity measured with substrate (Ac-IETD)2-R110
  • Compatible with both fluorometric and colorimetric detection systems
  • Fast enzyme kinetics
  • Enzymatic reaction forms yellow colored and green fluorescent R110 product
  • Endpoint assay requires cell lysis

Kit Components

  • Cell lysis buffer
  • 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.

Although fluorometric detection of the end products is preferred because of the superior sensitivity, detection by absorbance is also possible. In fact, the extinction coefficient of R110 is 10 times higher than that of p-nitroaniline (pNA), a dye commonly used in chromogenic substrates, making R110-based substrates significantly more sensitive than pNA-based substrates, even by colorimetric detection.

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.

References

1. Neurochem Res (2009) Aug;34(8):1410-6. doi: 10.1007/s11064-009-9927-6
2. J Psychiatr Res (2009) May;43(8):754-62 doi: 10.1016/j.jpsychires.2008.10.002.
3. Eur J Pharmacol (2009) May 21;610(1-3):99-105 doi: 10.1016/j.ejphar.2009.03.046
4. Fundam Clin Pharmacol (2010) Aug;24(4):445-50 doi: 10.1111/j.1472-8206.2009.00789.x
5. Asian Pac J Cancer Prev (2018) 19(4): 917–922 doi: 10.22034/APJCP.2018.19.4.917

 

You may also like…