Enzyme Assays

Introduction

Figure 1. Representative diagram of substrate and product migration.

1. Dilute recombinant mouse HS6ST3 (R&D Systems, Catalog # 5406-ST) to 2.67 µg/mL, 2 µg/mL,
   

Figure 1. Representative diagram of substrate and product migration.

1. Dilute recombinant mouse CHST7 (R&D Systems, Catalog # 5108-ST) to 20 µg/mL, 10 µg/mL, 5 µg/mL, and 2.5 µg/mL in Assay Buffer 1.
   

Figure 1. Representative diagram of substrate and product migration.

1. Dilute recombinant mouse CHST1 (R&D Systems, Catalog # 5355-ST) to 20 µg/mL, 10 µg/mL, 5 µg/mL, and 2.5 µg/mL in Assay Buffer 1.

1. Dilute recombinant mouse ACE-2 (R&D Systems, Catalog # 3437-ZN) to 0.5 µg/mL in ACE-2 assay buffer [75 mM Tris (Amresco, Catalog # 0826), 1 M NaCl (J.T. Baker, Catalog # 3624-07), pH 7.5].
2. Dilute the substrate, Mca-Y-V-A-D-A-P-K(Dnp)-OH (R&D Systems, Catalog # ES007) to 20 µM with ACE-2 assay buffer.
3. Load a plate into the plate reader.
4. Add 50 µL of enzyme to each well.

Introduction

1. Dilute recombinant human Meprin alpha Subunit (R&D Systems, Catalog # 3220-ZN) to 0.1 mg/mL in TCNB buffer [50 mM Tris, 150 mM NaCl, 10 mM CaCl₂ (Sigma, Catalog # C-5080), 0.05% Brij® 35, pH 7.5] containing 0.1 µg/mL Trypsin. Mix well by gently vortexing.
2. Incubate the mixture at 37° C for 3 hours in a heat block covered with foil. Stop trypsin activity by adding 1 mM AEBSF (R&D Systems, Catalog # EI001).

Figure 1. Representative diagram of substrate and product migration.

1. Dilute recombinant human HS6ST1 (R&D Systems, Catalog # 5057-ST) to 20 µg/mL, 10 µg/mL, 5 µg/mL, and 2.5 µg/mL in Assay Buffer 1.