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Beta-glucosyltransferase
Description
The Beta-glucosyltransferase (BGT) of T-even bacteriophages catalyses the transfer of glucose from UDP-glucose donor to genomic 5-hydroxymethylcytosine (5-hmC) bases. BGT glucosylates 5-hmC independently of DNA sequence and structural context. It can be used to assess the relative abundance of 5-hmC in genomic DNA using glucosyl-sensitive restriction enzymes (e.g. MspI, MspJI or GlaI) or labeling methods combined with immunodetection. Since our 5-hmC antibodies do not recognize glucosylated 5-hmC, BGT treatment is a perfect tool to check for the specificity during hMeDIP and dot blot.
| Beta-glucosyltransferase | AF-112-0100 | 100 rxns | 129 $ |  |
Features
• Isolated from a recombinant source
• Glucosylates to completion 5-hydroxymethylcytosine DNA bases
• Supplied with optimized 10x Reaction Buffer and UDP-Glucose donor
• Can be used to assess the relative abundance of 5-hmC in genomic DNA using glucosyl-sensitive restriction enzymes or labeling methods combined with immunodetection
• Ideal to control hMeDIP experiments and anti-5hmC dot blot assays
Figure 1
Mouse embryonic stem cells DNA was fragmented by sonication using the Diagenode Bioruptor® (size range: 100-500 bp). One µg of sheared mouse embryonic stem cells was mixed with the three controls (C, 5-mC and 5-hmC) from the hMeDIP kit. The DNA mixture was then treated enzymatically with (Gluc) or without (NT) β-glucosyltransferase as per the included reaction condition. After purification with the PrepEaseTM PCR Purification 96-well Plate kit (PN 78761, USB®), the reaction was recovered in 108 µl nuclease-free water and added to 12 µl of buffer H1 from the hMeDIP kit (Cat. No. AF-104-0016) The IP incubation mix was then heat denatured and hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit (Cat. No. AF-104-0016). This kit includes: the monoclonal rat antibody & Rat IgG (Cat. No. AF-105-0025). Finally qPCR using specific primer pairs for the unmethylated, methylated and hydroxymethylated control DNA sequences were performed. After glucosylation, the monoclonal rat antibody is not able to recognize hydroxymethylated epitopes.
Source: Purified from an E. coli strain, which carries a bacteriophage T4 β-glucosyltransferase overexpressing plasmid.
Storage conditions: Storage at -20°C is recommended. Since the 10x reaction buffer contains DTT, it is highly recommended to store it in small aliquotes at -20°C. For long-term storage, store both the enzyme and the 10 reaction buffer at -80°C. Avoid multiple freeze thawing. When stored properly the enzyme is stable for at least 6 months from date of receipt.
Package content:
• 1 tube of β-glucosyltransferase (enough for 100 Reactions)
• 1 tube of 600µl of 10 X β-glucosyltransferase reaction buffer (enough for 100 x 50µl assays)
• 1 tube of 1 mM UDP-glucose donor (enough for 100 x 50 µl assays)
Glucosylation of 5-hydroxymethylcytosine
Ideal to control hMeDIP and dot blot experiments
