Custom Premix Selecting "Custom Premix" option means that all of the beads you have chosen will be premixed in manufacturing before the kit is sent to you.
Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.
Revision
18-September-2008 RFH
Description
Substrate for fluorogenic assay of plasma and glandular kallikreins. Also serves as a substrate for cathepsin B, cathepsin L, and papain.
Form
White to off-white solid
Recommended reaction conditions
Protocol for the Assay of Cathepsin LEnzyme:
Cathepsin L is one of the lysosomal cysteine proteinases involved in the turnover of endocytosed proteins. It also has a higher specific activity towards physiological substrates, such as collagen and fibronectin over other lysosomal enzymes. It has an acidic optimal pH of 5.5. It is rapidly inactivated, 85% loss of activity in ~15 min, at pH 7.0-7.4.
Substrate:
Z-Phe-Arg-7-amido-4-methylcoumarin is more sensitive to cathepsin L than cathepsin B, although both enzymes will cleave the substrate. To determine cathepsin L activity, the specific cathepsin B substrate, Z-Arg-Arg-7-amido-4-methylcoumarin (Cat. No. 03-32-1570), can be used. The substrate is non-fluorescent, but when hydrolyzed by the enzyme it releases highly fluorescent 7-amido-4-methylcoumarin (AMC). AMC can be quantitated with a spectrofluorometer using an excitation at ~370 nm and an emission at ~460 nm.
Reagents:• Assay/Activator: 340 mM sodium acetate, 60 mM acetic acid, and 4 mM EDTA, Buffer pH 5.5; add DTT to a final concentration of 8 mM just prior to use.
• Substrate: Prepare a 1 mM Z-Phe-Arg-7-amido-4-methylcoumarin stock solution in DMSO and store in a -20°C freezer. Dilute to 20 µM with H2O prior to use.
• Stop Solution: 100 mM sodium monochloroacetate, 70 mM acetic acid, and 30 mM sodium acetate, pH 4.3
• Diluent: BRIJ® 35 (0.1% in H2O)
• Standard: Prepare a 1 mM AMC stock solution in DMSO and store in a 4°C refrigerator. Dilutions are made using a 1:1 mixture of Assay Buffer and Stop Solution.
Procedure:
1. Using the Diluent, prepare 500 µl of enzyme sample in 5 ml glass or polystyrene tubes.
2. Add 250 µl of assay/activator buffer. Allow ~1 min. incubation at 30°C for temperature equilibration and enzyme activation. Note: longer incubation may result in loss of activity.
3. Add 250 µl of the 20 µM substrate solution and mix.
4. Following a 10 min incubation at 30°C, add 1 ml of Stop Solution.
5. Measure the fluorescence and determine the molar amount using a standard curve for AMC. The fluorescence of AMC is only minimally affected by pH in the range of 4-7.
