Description
Green Cathepsin B Kit enables the quantitation and monitoring of intracellular cathepsin activity over time in vitro. The Rhodamine 110 Cathepsin B substrate reagent is a non-cytotoxic and membrane permeant substrate that fluoresces green upon cleavage by active cathepsin enzymes.
Test Principle: Rhodamine 110 Cathepsin B substrate utilizes the photostable green fluorophore, rhodamine 110. Rhodamine 110 cathepsin B substrate is comprised of rhodamine 110 coupled to two copies of the amino acid sequence, arginine-arginine (RR), which is the preferential target sequence for cathepsin B. When bi-substituted via amide linkage to two cathepsin B target peptide sequences, rhodamine110 is nonfluorescent. Following enzymatic cleavage at one or both arginine (R) amide linkage sites, the mono and non-substituted rhodamine 110 fluorophores generate green fluorescence when excited at 500 nm.
To use the Green Cathepsin B Assay, simply add the Rhodamine 110 Cathepsin B substrate [R110-(RR)2] directly to the cell culture media (or 1X Cellular Assay Buffer), incubate, and analyze. Because R110-(RR)2 is cell-permeant, it easily penetrates the cell membrane and the membranes of the internal cellular organelles - no lysis or permeabilization steps are required. R110-(RR)2 will enter the cell in a non-fluorescent state. If cathepsin enzymes are active, they will cleave off the two arginine-arginine cathepsin B targeting sequences and allow the rhodamine 110 fluorophore to become fluorescent upon excitation. By varying the duration and concentration of exposure to the R110-(RR)2 substrate, a picture can be obtained of the relative abundance and intracellular location of cathepsin enzymatic activity. Positive cells will fluoresce green, while negative cells will exhibit very low levels of