Skip to Content
Merck
  • Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads.

Pull-down of 5-hydroxymethylcytosine DNA using JBP1-coated magnetic beads.

Nature protocols (2012-01-28)
Adam B Robertson, John Arne Dahl, Rune Ougland, Arne Klungland
ABSTRACT

We describe a method for the efficient and selective identification of DNA containing the 5-hydroxymethylcytosine (5-hmC) modification. This protocol takes advantage of two proteins: T4 β-glucosyltransferase (β-gt), which converts 5-hmC to β-glucosyl-5-hmC (β-glu-5-hmC), and J-binding protein 1 (JBP1), which specifically recognizes and binds to β-glu-5-hmC. We describe the steps necessary to purify JBP1 and modify this protein such that it can be fixed to magnetic beads. Thereafter, we detail how to use the JBP1 magnetic beads to obtain DNA that is enriched with 5-hmC. This method is likely to produce results similar to those of other 5-hmC pull-down assays; however, all necessary components for the completion of this protocol are readily available or can be easily and rapidly synthesized using basic molecular biology techniques. This protocol can be completed in less than 2 weeks and allows the user to isolate 5-hmC-containing genomic DNA that is suitable for analysis by quantitative PCR (qPCR), sequencing, microarray and other molecular biology assays.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Manganese(II) chloride tetrahydrate, ACS reagent, ≥98%
Sign Into View Organizational & Contract Pricing
SKUPack SizeAvailabilityPriceQuantity
1 ea
Please contact Customer Service for Availability
HUF 94,200.00
Sigma-Aldrich
Sodium selenite, BioReagent, suitable for cell culture, ≥98%
Sign Into View Organizational & Contract Pricing
SKUPack SizeAvailabilityPriceQuantity
1 ea
Please contact Customer Service for Availability
HUF 94,200.00
Sigma-Aldrich
Copper(II) chloride dihydrate, powder, BioReagent, suitable for cell culture
Sign Into View Organizational & Contract Pricing
SKUPack SizeAvailabilityPriceQuantity
1 ea
Please contact Customer Service for Availability
HUF 94,200.00
Sigma-Aldrich
Proteinase K from Tritirachium album, lyophilized powder, BioUltra, ≥30 units/mg protein, for molecular biology
Sign Into View Organizational & Contract Pricing
SKUPack SizeAvailabilityPriceQuantity
1 ea
Please contact Customer Service for Availability
HUF 94,200.00
Sigma-Aldrich
Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder
Sign Into View Organizational & Contract Pricing
SKUPack SizeAvailabilityPriceQuantity
1 ea
Please contact Customer Service for Availability
HUF 94,200.00
Sigma-Aldrich
EZMix N-Z-Amine® A, Casein enzymatic hydrolysate, from bovine milk
Sign Into View Organizational & Contract Pricing
SKUPack SizeAvailabilityPriceQuantity
1 ea
Please contact Customer Service for Availability
HUF 94,200.00
Sigma-Aldrich
Albumin from porcine serum, lyophilized powder, essentially globulin free, ≥99% (agarose gel electrophoresis)
Sign Into View Organizational & Contract Pricing
SKUPack SizeAvailabilityPriceQuantity
1 ea
Please contact Customer Service for Availability
HUF 94,200.00