CATHEPSIN B K L APOPTOSIS CASPASE DETECTION KITS FOR IN VITRO USE IN REAL TIME MAGIC RED

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Cathepsin K Magic Red™ Real-Time Detection Kits
Visualize cathepsin K activity in whole living cells.

Cathepsin K-positive Jurkat cells fluoresce red after staining with MR-LR cat. #940
Cathepsin K-positive Jurkat cells fluoresce red after staining with MR-LR.

MR-Cathepsins Manual

Cathepsin K Magic Red™ kits contain: MR-LR reagent, Hoechst 33342, and acridine orange. 

Magic Red™ Cathepsin K Kits
# tests cat# price
25 939 $154
100 940 $389

 

 

Time 0 and 16 hours later. Monitor protease activity in real time. Read about this experiment

 

 

     ICT's Magic Red™-LR kits measure active cathepsin K in whole living, intact cells - no lysis required. As cathepsin K activity increases, the red fluorescent signal increases. These unique kits are not ELISAs and do not use antibodies - instead they use a cell-permeant substrate sequence, LR, specifically targeted by active cathepsin K. This peptide sequence is linked to a red fluorophore, Magic Red™, which fluoresces once cleaved by active cathepsin K.
     Only cells with active cathepsin K will fluoresce, so you don’t get any signal from pro-enzymes or inactive forms of the enzyme. ICT’s MR-LR kits give you a clear picture of cathepsin-K-positive versus negative cells.
     Cathepsin-K-positive cells often generate a signal 2-5x greater than the negative cell population when read in fluorescence plate readers. MR excites at 540-590 and emits at >610nm. This signal can be detected with a fluorescence microscope, a fluorescence plate reader, or special flow cytometers with adjustable excitation wavelengths. 
     MR kits are very specific. However, there still can be some cross-reactivity as the reagent is incubating (often for several hours). The Cathepsin K reagent has a high preference for the target sequence, MR-LR. Although Cathepsin K has its preferred amino acid sequence motif, it is still capable of reacting with other less desirable substrate sequences albeit at a lower efficiency in terms of kcat/Km ratios.
     In these cells we are seeing the hydrolysis of the MR-LR substrate, which has a preference for Cathepsin K. However, the reagent could by cleaved by other less desirable proteases albeit at a lower efficiency in terms of kcat/Km ratios. Cathepsin K is lysosomal. After staining with MR-LR, there are often areas of bright red fluorescence as the reagent is processed and concentrated within the lysosomal bodies.  

Some characteristics of cathepsin K:
1.  Limited tissue distribution. Found in osteoclasts and breast cancer carcinoma cells, bronchial epithelial cells, and in multinucleated and granuloma type cells.
2.  Associated with excessive bone resorption pathologies such as osteoporosis.
3.  Degrades type I collagen which is the main component of the bone matrix.
4.  An endopeptidase.

Find more details on the main MR page

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Frequently Asked Questions

 

Reference
1. Creasy, B., Hartmann, C., Higgins White, F., and McCoy, K. 2006. New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations. In press.

See all references (pdf)

 

Sample Protocol
1. Culture your cells individually or up to 1 x 106 cells/mL. 
2. Induce your experimental conditions following your protocol. 
3. Reconstitute the reagent with DMSO to form the stock concentrate (which can be frozen for future use). 
4. Dilute the stock concentrate with 1X PBS to form the working solution. 
5. Add ~10uL of the working solution directly to a 300-500uL aliquot of your cell culture for labeling. 
6. Incubate, typically 1-4 hours; protect cells from light.
7. If desired, label DNA with Hoechst stain. 
8. If desired, label lysosomes with Acridine Orange. 
9. If desired, fix cells. 
10. Analyze data using a fluorescence microscope, or plate reader.

References
Lee, B., G. L. Johnson, S. A. Hed, Z. Darzynkiewicz, J. Talhouk, and S. Mehrotra.  2001.  DEVDase detection in intact apoptotic cells using the cell permeant fluorogenic substrate, (z-DEVD)2-cresyl violet.  Biotechniques vol 35 no 5:1080-1085. Read this publication .

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Copyright 2008
04/04/2008 11:43 AM 		  Please contact Sally Hed with questions 952-888-8788 x10 sally@immunochemistry.com
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