A serine protease with caspase-like substrate specificity, Granzyme B is a component of the cytolytic granules of cytotoxic T lymphocytes and natural killer cells. This product is best to study enzyme regulation and kinetics, cleave target substrates, and screen for inhibitors.
Highly active Granzyme B expressed in yeast from a human cDNA. Study enzyme regulation and kinetics, cleave target substrates, and screen for inhibitors.
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1. Prepare Assay Buffer.
2. Dilute Granzyme B to 6 U/µL in Assay Buffer just before use.
3. Prepare 400 µM Ac-IEPD-pNA colorimetric substrate. If supplied as 400 µM stock solution in assay buffer, store at -20°C and warm to assay temperature before use. To prepare substrate from 5 mg net peptide (MW=639), add 156 µL DMSO to make a 50 mM stock. Dilute 50 mM stock to 400 µM with Assay Buffer.
4. Add 45 µL Assay Buffer into ½ volume microtiter plate. Allow to equilibrate to assay temperature.
5. Add 5 µL of Granzyme B (6 U/µL) to each appropriate well. Include 2 blanks (5 µL assay buffer rather than Granzyme B).
6. To start reaction, add 50 µL Ac-IEPD-pNA substrate (400 µM in assay buffer). Final substrate concentration = 200 µM.
7. Continuously monitor A450nm.
8. Data analysis: Graph OD vs time and determine slope over the linear portion of the curve. Convert rates in OD/min to substrate/min using an extinction coefficient for p-nitroaniline of 10,500 M-1 cm-1, and adjusting for pathlength of sample (~0.5 cm for 100 µL in well of a ½ volume, 96-well plate).
• Specific Activity: 700 U/µg. One unit hydrolyzes 1 pmol/min of Ac-IEPD-pNA (200 µM) at 30 °C
• Supplied As: 100 U/µL in 50 mM Tris, pH 7.4, 100 mM NaCl, 10% glycerol
• Storage: -70°C