APT139 | Caspase 9 Colorimetric Activity Assay Kit, LEHD

APT139
25 assays  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityKey ApplicationsDetection Methods
      MaACTChromogenic
      Description
      Catalogue NumberAPT139
      Brand Family Chemicon®
      Trade Name
      • Chemicon
      DescriptionCaspase 9 Colorimetric Activity Assay Kit, LEHD
      OverviewActivation of ICE-family proteases/caspases initiates apoptosis in mammalian cells. Chemicon's Caspase-9 Colorimetric Activity Assay Kits provide a simple and convenient means for assaying the activity of caspases that recognize the LEHD. The assay is based on spectophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate LEHD-pNA. The free pNA can be quantified using a spectrophotometer or a microtiter plate reader at 405 nm. Comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in caspase-9 activity.
      Alternate Names
      • Mch-6
      • ICE-LAP6
      • Apaf-3
      Materials Required but Not Delivered· Microcentrifuge and 1.5 mL Microcentrifuge tubes

      · 37°C Waterbath or Incubator

      · Spectrophotometer or Microplate Reader

      · 96 well Microtiter Plate
      References
      Product Information
      Components
      • 5X Cell Lysis Buffer (Part No. 90065): 5 mL
      • 5X Assay Buffer (Part No. 90066): 10 mL
      • Caspase-9 Substrate (Ac-LEHD-pNA) (Part No. 90083): 250 μL of 3 mg/mL solution
      • Caspase-9 Inhibitor (Ac-LEHD-CHO) (Part No. 90087): 50 μL of 100 μM (0.054 mg/mL) in DMSO
      • pNA Standard (Part No. 90085): 250 μL of 10 mM in DMSO
      Detection methodChromogenic
      Applications
      ApplicationCaspase-9 Colorimetric Activity Assay Kits provide a simple & convenient means for assaying the activity of caspases that recognize the LEHD.
      Key Applications
      • Activity Assay
      Biological Information
      Species Reactivity
      • Mammals
      Entrez Gene Number
      Entrez Gene SummaryThis gene encodes 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 is processed by caspase APAF1; this step is thought to be one of the earliest in the caspase activation cascade. Alternative splicing results in two transcript variants which encode different isoforms.
      Gene Symbol
      • CASP9
      • CASP-9
      • ICE-LAP6
      • APAF3
      • MCH6
      • CASPASE-9c
      • OTTHUMP00000044594
      • APAF-3
      • EC 3.4.22.62 [Contains: Caspase-9 subunit p35
      • Caspase-9 subunit p10].
      UniProt Number
      UniProt SummaryFUNCTION: SwissProt: P55211 # Isoform 2 lacks activity is an dominant-negative inhibitor of caspase-9.
      SIZE: 416 amino acids; 46281 Da
      SUBUNIT: Heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a 35 kDa (p35) and a 10 kDa (p10) subunit. Caspase-9 and APAF1 bind to each other via their respective NH2-terminal CED-3 homologous domains in the presence of cytochrome C and ATP. Interacts with the inhibitors BIRC2, BIRC4, BIRC5 and BIRC7.
      TISSUE SPECIFICITY: Ubiquitous, with highest expression in the heart, moderate expression in liver, skeletal muscle, and pancreas. Low levels in all other tissues.
      PTM: Cleavages at Asp-315 by granzyme B and at Asp-330 by caspase- 3 generate the two active subunits. Caspase-8 and -10 can also be involved in these processing events.
      SIMILARITY: SwissProt: P55211 ## Belongs to the peptidase C14 family. & Contains 1 CARD domain.
      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 ConditionsStore kit materials at -20°C up to their expiration date.

      Special Precautions:

      · After thawing reagents, use immediately or aliquot and freeze at -20°C for longer storage. Avoid repeated freeze/thaw cycles.

      · The Caspase-9 substrate and pNA standard are especially light sensitive. Maintain these reagents in amber or covered containers.
      Packaging Information
      Material Size25 assays
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      SDS

      Title

      Safety Data Sheet (SDS) 

      FAQ

      QuestionAnswer
      Why does caspase often exhibit different molecular weights?Full-length caspase 3 (pro-form) is 32kDa. Upon activation, caspase-3 is cleaved generating two smaller subunits of 17 kDa and 12 kDa. Other Caspases include Caspase 1 (proenzyme at 45kDa and subunit at 20kDa); Caspase 6 at 34kDa; Caspase 7 at 38kDa; Caspase 8 at 55kDa and Caspase 9 at 46-48kDa. It should be noted however that it can be difficult to detect the active fragments of many caspases because of their small size and biologically short life. The pro-forms are much more stable and readily detectable.

      User Guides

      Title
      Caspase-9 Colorimetric Activity Assay Kit