T4 β-Glucosyltransferase

T4 β-glucosyltransferase (T4 BGT) is a DNA-modifying enzyme encoded by bacteriophage T4 that catalyzes the transfer of glucose (Glc) from uridine diphosphoglucose (UDP-Glc) to 5-hydroxymethylcytosine (5-hmC) residues in double-stranded DNA, resulting in the formation of β-glucosyl-5- hydroxymethylcytosine.

The enzyme is available in 500 and 2,500 unit sizes at a concentration of 10 U/µL. The enzyme is supplied with a 10× Reaction Buffer.

Applications

• Glucosylation or immunodetection of  5-hmC DNA.
• Differentiation of 5-hmC from 5-mC.
• 5-hmC containing DNA enrichment.
• Labeling of 5-hmC residues using a radioactive UDP-glucose donor.

Product Specifications

• Storage Buffer: 50 mM Tris-HCl (pH 7.5 at 25 ^oC), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, and 50% (v/v) glycerol.
• Unit Definition: One unit is defined as the amount of enzyme required to protect 0.5 µg T4gt-DNA against cleavage by MfeI restriction endonuclease.
• Protection Unit Assay Conditions: 0.5 µg T4gt-DNA, 1X reaction buffer and 40 µM UDP-Glucose in a 30 µL reaction. Incubate for 1 hour at 37°C followed by 10 minutes at 65°C. The extent of protection by T4 β-glucosyltransferase is determined by the addition of 20 µL 1X reaction buffer and 10 units of MfeI. Incubation at 37°C for 30 minutes is followed by analysis on agarose gels.

General Protocol

 1. Assemble the following reaction at room temperature:

         10× Reaction Buffer                  5 µL
         2 mM UDP-Glucose                  5 µL
         DNA                                          up to 1 µg
         Nuclease-free water                  to 49 µL
        T4 BGT                                     1 µL
        Total volume                             50 µL

2. Mix gently and spin down for a few seconds.

3. Incubate at 37 °C for 1 hour.

4. Stop the reaction by heating at 65 °C for 20 min.