Molecular Techniques and Methods

In vitro Assay of UDP-Glycosyltransferase in Microsomes

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS




PROCEDURES

1. Make the 50 ul enzyme reaction mixture as follow:

UGT in Microsomes ------------------------------------------- 100 ug/ 5 ul
100 mM Tris-HCl (pH 7.0) ----------------------------------- 2.5 ul of 2 M Tris-HCl
10 mM MgCl2 -------------------------------------------------- 1 ul of 0.5 M MgCl2
6.8 uM UDP-D-[U-14C]-glucuronic acid -------------------- 0.5-1 uCi in 5-10 ul
Acceptor Substrate in 10% DMSO ---------------------------- 5 ul
Distilled H2O ---------------------------------------------------- 31.5 ul


2. Incubate 30 min - 2 hours at 25-30oC.

3. Stop reaction by adding 50 ul Me-OH.

4. Analyze the reaction products on silica gel 60-TLCF254 plate.
  • Ex) Acetonitrile: Acetone: iPr-OH: Acetic acid: H2O = 5:8:10:1:2



    NOTES



    KIT INFORMATION



    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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