Apoptosis

Supplied by B-Bridge International, Inc

Metachem Diagnostics are delighted to be the UK distributors for B-Bridge International, Inc.  B-Bridge International supply the first ELISA kits for the measurement of Adiponectin and a wide range kits for the investigation of Apoptosis

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Introduction

Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialised 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.

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 the upstream regulatory events (initiator caspases).  An active caspase consists of two large (-20 kDa) and two small (-10 kDa) subunits that form two heterodimers which associate in a tetramer.  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 recognise a 4 amino acid sequence (on the target substrate) that necessarily includes an aspartic acid residue.  The residue is the target for the cleavage reaction, 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 that become fluorescent upon cleavage by the caspases. 

FLICA Apoptosis Detection Kits use a novel approach to detect active caspases.  The methodology is based on a Fluorochrome Inhibitor of Caspase (FLICA).  Once inside the cell the FLICA inhibitor binds covalently to the active caspase 7.  These inhibitors are cell permeable and non -cytotoxic.  Fpr kits using green fluorescence a carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor of caspases is used.  A line of red FLICA Apoptosis Detection Kits that use sulforhodamine-labeled inhibitors is also available.

For example, the Poly-Caspases FLICA Apoptosis Detection Kit contains a green fluorescent-labeled inhibitor, FAM-VAD-FMK, which is a carboxyfluorescein (FAM) derivative of valylalanylaspartic acid (VAD) fluoromethyl ketone (FMK), a potent inhibitor of caspase activity.  When added to a population of cells, the FAM-VAD-FMK FLICA probe enters each cell and covalently binds to the reactive cysteine residue that resides on the large subunit of the active caspase heterodimer, thereby inhibiting further enzymatic activity.  Because the FAM-VAD-FMK FLICA reagent becomes covalently coupled to the enzyme, it is retained within the cell, while any unbound FAM-VAD-FMK FLICA reagent will diffuse out of the cell and is washed away.  The remaining green fluorescent signal is a direct measure of the number of active caspase enzymes that were present at the time the reagent was added.  Cell that cotain the bound FLICA can be analyzed by 96 well-plate based fluorometry, fluorescence microscopy or flow cytometry.

Because the FLICA reagent FAM-VAD-FMK irreversibly binds to many activated caspases (caspase-1, -3, -4, -5, -6, -7, -8, and -9) it can be used as a generic probe for the detection of most caspases.  In comparison, when FLICA reagent FAM-YVAD-FMK enters the cell, it primarily binds to caspase-1, therefore it can be used to measure the amount of active caspase-1 that was present in the cell at the time when the FAM-YVAD-FMK FLICA reagent was added.  Other green fluorescence FLICA Apoptosis Detection Kits are available to analyse specific caspases: FAM-VDVAD-FMK FLICA can be used to detect caspase-2, FAM-DEVD-FMK to detect caspase-3 and caspase-7, FAM-VEID-FMK to detect caspase-6, FAM-LETD-FMK to primarily detect caspase-8, FAM-LEHD-FMK to primarily detect caspase-9 FAM-AEVD-FMK to detect caspase-10 and FAM-LEED-FMK to detect primarily caspase-13.

Please click on hyperlinks below for product details

Carboxyfluorescein FLICA Assay Kits

Sulforhodamine FLICA Assay Kits

Mitochondrial Permeability Transition Detection Kits

Magic Red Cathepsin Detection Kits

Immunoassay Kits

 

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