Jul 17, 2020
Version 2
Enzymatic Assay of Protease Using Azocasein as Substrate V.2
- 1University of Manitoba
Protocol Citation: Neilier Junior 2020. Enzymatic Assay of Protease Using Azocasein as Substrate. protocols.io https://dx.doi.org/10.17504/protocols.io.bhqnj5ve
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: June 19, 2020
Last Modified: July 17, 2020
Protocol Integer ID: 38382
Keywords: Enzyme, substrate, kinetic, enzymology, protein
Materials
MATERIALS
Calcium chloride Merck MilliporeCatalog #1.02378.0500
Trichloroacetic acid (TCA)Sigma – AldrichCatalog #T6399
Sodium hydroxideSigma – AldrichCatalog #S8045
Trizma® base Sigma AldrichCatalog #T4661
AzocaseinCatalog #A2765
Safety warnings
Wear personal protective equipment: gloves, lab coat and mask.
Before start
Organize your workspace
Make sure all solutions and equipment are available.
Reagent Preparation
Reagent Preparation
- 100 mM Tris-HCl buffer, pH 8.0, 20 mM CaCl2, at 37 °C.
- 2.0% (w/v) Azocasein Solution
Heat gently (do not boil) to 50 - 60 °C for 10 min with stirring.
Adjust the pH to 8.0 at 37 °C, if necessary, with either 1.0 M NaOH or 1.0 M HCl.
- 110 mM Trichloroacetic Acid Reagent (TCA). Dilute with deionized water.
- 500 mM Sodium Hydroxide (NaOH) Solution. Prepare in deionized water.
Check how many samples will be analyzed to calculate the required volume of each solution to be prepared.
Procedure
Procedure
Pipette (in microliters) the following reagents into 2.0 mL microtubes.
| Blank | Test | ||
| Tris-HCl buffer | 750 μL | 450 μL | |
| Azocasein | 750 μL | 750 μL | |
| Mix and equilibrate to the at desired temperature. Then add: | * | ||
| Sample (enzyme source) | - | 300 μL | |
| Mix and incubate at desired temperature for exactly 30 min. | * | ||
| Remove a 1 mL aliquot from both (test and blank) solutions and place into 2.0 mL microtubes. Then add: | |||
| TCA | 1000 μL | 1000 μL | |
| Centrifuge at 20,000 g for 10 min. Remove a 1 mL aliquot from supernatant (test and blank) and place into 2.0 mL microtubes. Then add: | * | ||
| NaOH | 1000 μL | 1000 μL | |
| Mix and transfer the Test and Blank solutions to suitable cuvettes. Measure the A440nm for Test and Blank using a spectrophotometer. | * | ||
Calculation
Calculation
ΔA440nm = A440nmTest - A440nmBlank