酶活性分析实验指南
Enzyme Assays
Sulfotransferase Activity Assays
Introduction
Recombinant Mouse Heparan Sulfate 6-O-Sulfotransferase-3/HS6ST3 Activity Assay
1.Dilute recombinant mouse HS6ST3 (R&D Systems, Catalog # 5406-ST) to 2.67 µg/mL, 2 µg/mL,
Recombinant Mouse Carbohydrate Sulfotransferase 7/CHST7 Activity Assay
Figure 1. Representative diagram of substrate and product migration.
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.
Recombinant Mouse Carbohydrate Sulfotransferase 5/CHST5 Activity Assay
Recombinant Mouse Carbohydrate Sulfotransferase 1/CHST1 Activity Assay
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.
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].
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.
Load a plate into the plate reader.
Add 50 µL of enzyme to each well.
Add 50 µL of substrate to each well.
Inhibition of Calcineurin Phosphatase Activity
Introduction
Recombinant Human Meprin alpha Subunit/MEP1A Assay
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.
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).
Recombinant Human Sulfate 6-O-Sulfotransferase 1/HS6ST1 Activity Assay
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.
Recombinant Human GalNAc4S-6ST/BRAG Activity Assay
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