Magic Red TM Cathepsin Detection Kits

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Elevated cathepsin enzyme activity in serum or the extracellular matrix often signifies a number of gross pathological conditions.  Cathepsin-mediated diseases include: Alzheimer's, numerous types of cancer, autoimmune related diseases like arthritis and the accelerated breakdown of bone structure seen with osteoporosis.  Up-regulated cathepsin B and L activity has been linked to several types of cancer.  These include cancer of the colon, pancreas, ovaries, breast, lung and skin (melanoma).  Up-regulation of cathepsin K has been shown un lung tumors.  Increased cathepsin K activity has also been linked to degenerative bone diseases including osteoporosis and post-menopausal osteoporosis.  

Cathepsins are usually characterised as members of the lysosomal cysteine protease (active site) family and the cathepsin family name has been synonymous with lysosomal proteolytic enzymes.  In actuality, the cathepsin family also contains members of the serine protease (cathepsin A,G) and aspartic protease (cathepsin D,E) families as well.  These enzymes exist in their processed form as disulfide-linked heavy and light chain subunits with molecular weights ranging from 20-35 kDa.  Cathepsin C is the noted exception, existing as an oligomeric enzyme with a MW -200kDa.  Initially synthesized as inactive zymogens, they are post-transiationally processed into their active configurations after passing through the endoplasmic reticulum and subsequent incorporation into the acidic environment of the lysosomes. 

Protease activity can be detected within whole living cells using Magic Red TM substrate-based assay kits.  Designated as the MR-Cathepsin product line, these kits allow researchers to quickly visualise intracellular cathepsin activity within their particular experimental cell line.

The Magic Red TM detection kits utilise the fluorophore, cresyl violet.  When bi-substituted via amide linkage to two cathepsin target sequence peptides {such as (Aginine-Arginine)2 }, the cresyl violet leaving group is non-fluorescent.  Following enzymatic cleavage at one or both arginine (R) amide linkage sites, the mono and non-substituted cresyl violet fluorophores generate red fluorescence when excited at 550-590 nm.

In the Magic Red TM Cathepsin Detection Assay Systems, (x-Arginine-Arginine)2 abbreviated as (z-RR)2 (z-leucine-Arginine)2 abbreviated as (z-LR)2 and (z-Phenylalanine-Arginine)2 abbreviate as (z-FR)2 derivatives of the cresyl violet fluorophore (synthesized by Enzyme Systems Products) have been incorporated into user-friendly assay kits.  These MR-Cathepsin photostable fluorogenic substrates easily penetrate the cell membrane and the membranes of the internal cellular organelles enabling  researchers to detect cathepsin activity within whole living cells.

Intracellular cresyl violet substrate hydrolysis can be monitored by the accumulation of red fluorescent product within various organelles.  By varying the duration and concentration of exposure to the MR-Cathepsin substrate, a picture can be obtained of the relative abundance and intracellular location of cathepsin enzyme activity.  This activity can be monitored using a fluorescence microscope or microtitre plate fluorometer.

Hoechst stain is included and can be used to label the cell nuclei after labeling with the MR-Cathepsin reagent.  In addition, acridine orange (AO) is included in the kit to help identify lysosomes and other intracellular organelles.  The acidic pH of the lysosome results in the concentration and aggregation of the AO molecules.  Aggregated AO molecules fluoresce orange rather than green thus clearly differentiating the lysosomes from the other organelles.

 

Detection of intracellular cathepsin L activity in Jurkat cells using (z-FR)2
Photo A : Cathepsin fluorogenic substrate. Localisation of the hydrolysed (fluorescent) Magic Red TM Photo B : shows the corresponding differential interference contrast (DIC)image of the cells


                                                                                                                                                            

Magic Red TM Detection Kits

Product Peptide  Size Catalogue Number
Caspase 3 & 7 MR-(DEVD)2 25 tests BB935
Caspase 3 & 7 MR-(DEVD)2 100 tests BB936
Cathepsin B MR-(RR)2 25 tests BB937
Cathepsin B MR-(RR)2 100 tests BB938
Cathepsin K MR-(LR)2 25 testd BB939
Cathepsin K MR-(LR)2 100 tests BB940
Cathepsin L MR-(FR)2 25 tests BB941
Cathepsin L MR-(FR)2 100 tests BB942

                                 

                             For Research use only

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