Magic Red™ Cathepsin B Detection Kit
Assess cathepsin B activity in vitro and in real time
ICT's Magic Red™-RR kits measure active cathepsin B in whole living, intact cells - no lysis required. As cathepsin B 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, RR, specifically targeted by active cathepsin B. This peptide sequence is linked to a red fluorophore, Magic Red™, which fluoresces once cleaved by active cathepsin B. Only cells with active cathepsin B will fluoresce, so you don’t get any signal from pro-enzymes or inactive forms of the enzyme. ICT’s MR-RR kits give you a clear picture of cathepsin-B-positive versus negative cells.
Cathepsin-B-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). ICT's Cathepsin B (MR-RR) reagent is the most specific of the cathepsin reagents - there is very little cross reactivity with other proteases. Cathepsin B is highly abundant and highly active.
Cathepsin B is thought to be the most abundant cathepsin in the cell. It is lysosomal. After staining with MR, there are often areas of bright red fluorescence as the reagent is processed and concentrated within the lysosomal bodies.
Some characteristics of cathepsin B:
1. A endopeptidase with carboxydipeptidase activity.
2. An abundant lysosomal protease like L and D.
3. Associated with cancer malignancy.
4. Involved in breakdown of proteins after phagocytosis and in subsequent antigen presentation.
5. Requires an acidic environment for enzymatic activity.
Magic Red Cathepsin B Detection kit contains:
- ICT's red Magic Red cathepsin B detection probe, MR-(RR)2
- Hoechst stain (a blue DNA stain)
- Acridine orange (a lysosomal stain)
- MR-Cathepsins Manual
Trial size, ~25 tests:
Regular size, ~100 tests:
Learn more at ICT's version 1.0 Cathepsin B webpage
Cathepsin B-positive THP-1 cells fluoresce red after staining with MR-RR. Red fluorophore concentrates inside lysosomes.
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.