APT400 | CaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein

APT400
100 assays  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityKey ApplicationsDetection Methods
      MaFC, ACTFluorescent
      Description
      Catalogue NumberAPT400
      Brand Family Chemicon®
      Trade Name
      • CaspaTag
      • Chemicon
      DescriptionCaspaTag Pan-Caspase In Situ Assay Kit, Fluorescein
      OverviewApoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes 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 (Slee et al. 1999).

      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 tetramer (Walker et al. 1994; Wilson et al. 1994; Rotonda et al. 1996). 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 (Kumar 1999).

      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 (Thornberry et al. 1997). 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.

      Test Principle:

      CHEMICON'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 (Ekert et al. 1999). This 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.

      Application:

      The CHEMICON In Situ FLICA Pan-Caspase Detection Kit is a fluorescent-based assay for detection of active caspases in cells undergoing apoptosis. The kit is for research use only. Not for use in diagnostic or therapeutic procedures.
      Materials Required but Not Delivered· Cultured cells with media

      · Reagents to induce apoptosis

      · 15 mL polystyrene centrifuge tubes

      · Amber vials or tubes for storage of 150X concentrate at -20°C

      · 600 mL graduated cylinder

      · Microscope slides

      · Hemocytometer

      · Centrifuge (400 x g)

      · 37°C incubator

      · Vortexer

      · Adjustable volume pipettor with disposable tips

      · Deionized water

      · PBS, pH 7.4

      · DMSO
      References
      Product Information
      Components
      • FLICA Reagent (FAM-VAD-FMK): Four lyophilized vials
      • 10X Wash Buffer: 60 mL
      • Fixative (10% formaldehyde): 6 mL
      • Propidium Iodide: 1 mL at 250 μg/mL, ready-to-use
      • Hoechst Stain: 1 mL at 200 μg/mL, ready-to-use
      Detection methodFluorescent
      Applications
      ApplicationThe In Situ FLICA Pan-Caspase Detection Kit for flow cytometry is a fluorescent-based assay for detection of active caspases in cells undergoing apoptosis.
      Key Applications
      • Flow Cytometry
      • Activity Assay
      Biological Information
      Species Reactivity
      • Mammals
      Entrez Gene Number
      Entrez Gene SummaryThis gene encodes a protein which is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes which undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This protein cleaves and activates caspases 3 and 7, and the protein itself is processed by caspase 8. Mutations in this gene are associated with apoptosis defects seen in type II autoimmune lymphoproliferative syndrome. Three alternatively spliced transcript variants encoding different isoforms have been described for this gene.
      Gene Symbol
      • CASP1
      • IL1BCE
      • P45
      • IL-1BC
      • IL1BC
      • p45
      • IL1B-convertase
      • CASP-1
      • ICE
      • EC 3.4.22.36 [Contains: Caspase-1 subunit p20
      • Caspase-1 subunit p10].
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: Q92851 # Isoform C is proteolytically inactive.
      SIZE: 521 amino acids; 58951 Da
      SUBUNIT: Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 23/17 kDa (p23/17) (depending on the splicing events) and a 12 kDa (p12) subunit (By similarity). Self-associates. Interacts with FADD and CASP8. Found in a Fas signaling complex consisting of FAS, FADD, CASP8 and CASP10.
      TISSUE SPECIFICITY: Detectable in most tissues. Lowest expression is seen in brain, kidney, prostate, testis and colon.
      PTM: Cleavage by granzyme B and autocatalytic activity generate the two active subunits. & Phosphorylated upon DNA damage, probably by ATM or ATR.
      DISEASE: SwissProt: Q92851 # Defects in CASP10 are the cause of autoimmune lymphoproliferative syndrome type 2A (ALPS2A) [MIM:603909]. ALPS2 is characterized by abnormal lymphocyte and dendritic cell homeostasis and immune regulatory defects. & Defects in CASP10 are a cause of familial non-Hodgkin lymphoma (NHL) [MIM:605027]. NHL is a cancer that starts in cells of the lymph system, which is part of the body's immune system. NHLs can occur at any age and are often marked by enlarged lymph nodes, fever and weight loss. & Defects in CASP10 are a cause of gastric cancers [MIM:137215].
      SIMILARITY: SwissProt: Q92851 ## Belongs to the peptidase C14 family. & Contains 2 DED (death effector) domains.
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Usage Statement
      • Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
      Storage and Shipping Information
      Storage Conditions· Store unopened kit materials at 2-8°C up to their expiration date.

      · Reconstituted FLICA Reagent (150X) should be frozen at -20ºC 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 -20ºC for 2 weeks.
      Packaging Information
      Material Size100 assays
      Transport Information
      Supplemental Information
      Specifications

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      Categories

      Life Science Research > Protein Detection and Quantification > Activity Assays > Caspase & Apoptosis Detection