Description
Description: Magic Red Cathepsin B detection kits provide a simple method to monitor the varying constitutive levels of generalized cathepsin B enzyme activity in whole, living cells. Our Magic Red Cathepsin B assay is based on the cresyl violet (CV) fluorogenic substrate CV-(Arg-Arg)2 or CV-(RR)2. This cathepsin B-preferred peptide targeting sequence enables the detection of cathepsin B enzyme activity in whole, living, and intact cells. When the fluorophore is di-substituted with two R-R dipeptide cathepsin B targeting sequences, it is essentially non-fluorescent. Active cathepsin B lysosomal enzyme, present in the experimental cell population, will catalyze the hydrolysis of the two R-R target sequences from the cresyl violet fluorophore (aka Magic Red). Following cathepsin B-associated enzymatic hydrolysis, the two R-R peptide sequences are cleaved from the Magic Red molecule, converting it to the red fluorescent form. As cathepsin activity progresses, more of the MR-(RR)2 is converted to the red fluorescent form of the substrate, leading to an overall increase in the red fluorescence of the cell. Because the Magic Red probe is cell membrane permeant, a cell lysis step is not required to perform the assay. Magic Red excites at 540-590 nm (590 nm optimal) and emits at >610nm (630 nm optimal). This signal can be detected with a fluorescence microscope or flow cytometers equipped with a green excitation laser. Each kit includes the Magic Red-(RR)2 substrate reagent, Hoechst 33342 stain, and Acridine Orange stain, allowing for cells to be simultaneously evaluated for both the presence of apoptotic nuclear morphology and cathepsin B activity in fluorescence microscopy analysis techniques. Acridine Orange dye is included to visualize lysosomal organelle structure.
Background: Cathepsin B is a lysosomal cysteine protease and a member of the papain superfamily of enzymes. Cathepsin B functions as a carboxydipeptidase, demonstrating both endo and exopeptidase activity in its degradation and processing of a wide range of protein substrates [1]. In addition to its lysosomal role as a processor of intracellular proteins, its involvement as a mediator of tumor metastasis and degenerative diseases including Alzheimer's Disease has made this enzyme an important research topic [2-4]. Assessment of elevated levels of extracellular cathepsins has proven to be an important prognostic indicator of cancer chemotherapy outcome [4]. Magic Red Cathepsin B detection kits provide a simple method to monitor the varying constitutive levels of generalized cathepsin B enzyme activity in whole, living cells