Jan 11, 2026
Magic Red® Cathepsin B Assay Protocol
- Ali Ghoochani1,2,3,4,
- Monther Abu-Remaileh1,2,3,4
- 1Department of Chemical Engineering, Stanford University, Stanford, CA 94305, USA;
- 2Department of Genetics, Stanford University, Stanford, CA 94305, USA;
- 3The Institute for Chemistry, Engineering and Medicine for Human Health (Sarafan ChEM-H), Stanford University, Stanford, CA 94305, USA;
- 4Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
- asap
Protocol Citation: Ali Ghoochani, Monther Abu-Remaileh 2026. Magic Red® Cathepsin B Assay Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32mb1v25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 13, 2024
Last Modified: January 11, 2026
Protocol Integer ID: 109768
Keywords: red fluorescent signal, magic red substrate, based assay, fluorophore cresyl violet, fluorescence microscope, fluorescence, cresyl violet fluorophore, immunochemistry technology, assay
Abstract
The Magic Red Cathepsin B Assay Kit from ImmunoChemistry Technologies is a fluorescence-based assay designed to detect and measure intracellular cathepsin B activity in live cells. This kit utilizes the Magic Red substrate (MR-RR2), which contains the fluorophore cresyl violet conjugated to a cathepsin B-specific dipeptide sequence (arginine-arginine). Upon cleavage by active cathepsin B, the substrate releases the cresyl violet fluorophore, resulting in a red fluorescent signal that can be detected using a fluorescence microscope or plate reader.
Materials
Magic Red Cathepsin B Assay Kit (ImmunoChemistry Technologies, Cat# 937.
Troubleshooting
Prepare Magic Red Staining Solution
Reconstitute Magic Red Substrate (MR-RR2) with 50 μL DMSO.
Add Staining Solution to Cells
Once cells are ready for the experiment:
Dilute the stock 1:10 with diH2O to form the staining solution.
Dilute the Magic Red staining solution 1:25. Add 20 μL staining solution to 480 μL media.
Incubate the cells with the Magic Red staining solution for 00:30:00 at 37 °C
30m
Imaging
Immediately image the cells using a fluorescent or confocal microscope.
Use filters appropriate for Magic Red detection (excitation: 550-590 nm, emission: >610 nm)
Note: If the background fluorescence appears high or if there is excess staining solution, a gentle PBS wash can be considered to reduce background.