Private workspaceEnzymatic Assay of Protease Using Azocasein as Substrate V.1

DOI
dx.doi.org/10.17504/protocols.io.bayzifx6
  • Neilier Junior1,
  • Rafael de Almeida Barros2,
  • Camilo Elber Vital2,
  • Samuel Lessa Barbosa2,
  • João Vitor Aguilar de Oliveira2,
  • Gabriele Corrêa da Rocha2,
  • Cauê Neves Oliveira2,
  • João Victor Marques Gonçalves Assis2
  • 1University of Manitoba;
  • 2Universidade Federal de Viçosa
  • Biochemistry Protocols
    Tech. support email: neilier.junior@ufv.br
Neilier Junior
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DOI: dx.doi.org/10.17504/protocols.io.bayzifx6
Protocol Citation: Neilier Junior, Rafael de Almeida Barros, Camilo Elber Vital, Samuel Lessa Barbosa, João Vitor Aguilar de Oliveira, Gabriele Corrêa da Rocha, Cauê Neves Oliveira, João Victor Marques Gonçalves Assis . Enzymatic Assay of Protease Using Azocasein as Substrate. protocols.io https://dx.doi.org/10.17504/protocols.io.bayzifx6
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
Created: January 04, 2020
Last Modified: January 06, 2020
Protocol Integer ID: 31481
Keywords: Enzyme, substrate, kinetic, enzymology, protein
Materials
MATERIALS
ReagentCalcium chloride Merck MilliporeCatalog #1.02378.0500
ReagentTrichloroacetic acid (TCA)Sigma – AldrichCatalog #T6399
ReagentSodium hydroxideSigma – AldrichCatalog #S8045
ReagentTrizma® base Sigma AldrichCatalog #T4661
ReagentAzocaseinCatalog #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.

BlankTest
Tris-HCl buffer750 μL450 μL
Azocasein750 μL750 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:
TCA1000 μL1000 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:*
NaOH1000 μL1000 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