APT428
CaspaTag Caspase 8 In Situ Assay Kit 25, Fluorescein
The CaspaTag Caspase-8 In Situ Assay Kit, Fluorescein for flow cytometry is a fluorescent-based assay for detection of active caspase-8 in cells undergoing apoptosis.
About This Item
Doporučené produkty
Quality Level
species reactivity (predicted by homology)
mammals
manufacturer/tradename
CaspaTag
Chemicon®
technique(s)
activity assay: suitable
flow cytometry: suitable
NCBI accession no.
UniProt accession no.
detection method
fluorometric
shipped in
wet ice
General description
Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large and two small subunits that form two heterodimers, which associate in a tetramer2-4. In common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity5.
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 residue6. 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.
Application
Apoptosis & Cancer
Test Principle
CHEMICON′s CaspaTag Caspase-8 In Situ Assay 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 caspase7. This kit uses a carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor of caspase-8 (FAM-LETD-FMK), which produces a green fluorescence. When added to a population of cells, the FAM-LETD-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-8 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.
Components
10X Wash Buffer: 15 mL
Fixative: 6 mL
Propidium Iodide: 1 mL at 250 μg/mL, ready-to-use
Hoechst Stain: 1 mL at 200 μg/mL, ready-to-use
Storage and Stability
· Reconstituted FLICA Reagent (150X) should be frozen at -20°C for up to 6 months, and may be thawed twice during this time. Aliquot into separate amber tubes if desired. Protect from light at all times.
· Store diluted (1X) wash buffer up to -20°C for 2 weeks.
Legal Information
Disclaimer
signalword
Danger
Hazard Classifications
Acute Tox. 3 Inhalation - Acute Tox. 4 Dermal - Acute Tox. 4 Oral - Carc. 1B - Eye Irrit. 2 - Muta. 2 - Skin Irrit. 2 - Skin Sens. 1 - STOT SE 2 - STOT SE 3
target_organs
Eyes,Central nervous system, Respiratory system
Storage Class
6.1C - Combustible acute toxic Cat.3 / toxic compounds or compounds which causing chronic effects
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