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Investigación en Ciencias de la Vida
 

Caspase Antibodies and Assays

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Caspase cascade.
A central component of the apoptotic process is a cascade of proteolytic enzymes called caspases. Caspases participate in a series of reactions that are triggered in response to pro-apoptotic signals and result in the cleavage of protein substrates, causing the disassembly of the cell. Caspases have been identified in organisms ranging from C. elegans to humans. In common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity.

Caspase enzymes specifically recognize a 4 or 5 amino acid sequence on the target substrate which necessarily includes an aspartic acid residue. This residue is the target for cleavage, which occurs at the carbonyl end of the aspartic acid residue. Caspases can be detected via immunoprecipitation, immunoblotting techniques using caspase specific antibodies, or by employing fluorochrome substrates which become fluorescent upon cleavage by the caspase.

Merck provides a wide range of caspase antibodies, inhibitors, proteins and assays to help detect and study caspase activation in apoptotic cells.

Product Highlight:
CaspaTag™ Pan-Caspase In Situ Assay Kit, Fluorescein
Caspase activity was detected as a fluorescent signal in the apoptotic cells.
Suspension cells were incubated with staurosporine to induce apoptosis. Cells were then labeled with the FAM-VAD-FMK probe. Caspase activity was detected as a fluorescent signal in the apoptotic cells. Non-apoptotic cells were not visible under fluorescence. Caspase activity in top photo was detected in one cell using a band pass filter. The second cell is not visible and therefore not apoptotic. Nuclear staining in bottom photo was revealed using a UV filter.
Merck's In Situ Caspase Detection Kits use a novel approach to detect active caspases. The methodology is based on Fluorochrome Inhibitors of Caspases (FLICA). The inhibitors are cell permeable and non-cytotoxic. Once inside the cell, the inhibitor binds covalently to the active caspase.

The CaspaTag Pan-Caspase In Situ Fluorescein Assay Kit uses a carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor of caspase (FAM-VAD-FMK), which produces a green fluorescence. When added to a population of cells, the FAM-VAD-FMK probe enters each cell and covalently binds to a reactive cysteine residue that resides on the large subunit of the active caspase heterodimer, thereby inhibiting further enzymatic activity. The bound labeled reagent is retained within the cell, while any unbound reagent will diffuse out of the cell and is washed away. The green fluorescent signal is a direct measure of the amount of active caspase present in the cell at the time the reagent was added. Cells that contain the bound labeled reagent can be analyzed by 96-well plate-based fluorometry, fluorescence microscopy, or flow cytometry.

Product Highlight:
CaspScreen Caspase Detection Kit, non-adherent cells only
Detection of apoptotic cells by CaspSCREEN Flow Cytometric Analysis Kit.
Detection of apoptotic cells by CaspSCREEN™ Flow Cytometric Analysis Kit
The CaspSCREEN Flow Cytometric Apoptosis Detection Kit provides a simple and convenient means for detecting activation of caspases by flow cytometry in intact cells. The assay utilizes a substrate that contains two aspartate residues linked to rhodamine 110 (D2R), a reported substrate for members of capase family proteases. The rhodamine 110 conjugated substrate is non-fluorescent, however, upon cleavage of the substrate by cellular caspases or apoptosis-related proteases, the released rhodamine 110 gives rise to fluorescence that can be measured at excitation of 488 nm and emission of 530 nm. As the D2R substrate is more cell-permeable than other fluorometric caspase substrates, apoptosis can easily be measured in intact cells by flow cytometry.


Merck's Family of Caspase Detection Assays

DescriptionCatalog No.Assays
CaspSCREEN, non-adherent cells only APT105 25 assays
CaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein APT400 100 assays
CaspaTag Caspase 3,7 In Situ Assay Kit, Fluorescein APT403 100 assays
CaspaTag Caspase 8 In Situ Assay Kit, Fluorescein APT408 100 assays
CaspaTag Caspase 9 In Situ Assay Kit, Fluorescein APT409 100 assays
CaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein APT420 25 assays
CaspaTag Caspase 3,7 In Situ Assay Kit, Fluorescein APT423 25 assays
CaspaTag Caspase 8 In Situ Assay Kit 25, Fluorescein APT428 25 assays
CaspaTag Caspase 9 In Situ Assay Kit, Fluorescein APT429 25 assays

Whether cells are in the early stages of apoptosis (with exposed inner leaflets), intermediate stages (with active caspase activity), or late stages (with extensive DNA fragmentation), Merck’s reagents and cell-based assays detect specific cell death processes across the apoptotic timeline.