MethylCollector™ MBD Capture Kit

fast magnetic assay for enrichment of CpG-methylated DNA

 

MethylCollector™ MBD Capture Kit Overview

Active Motif’s MethylCollector™ MBD Capture Kit provides an efficient method for isolating CpG-methylated DNA from limited amounts of cell or tissue samples.

MethylCollector has many powerful applications including enabling researchers to rapidly screen the methylation status of multiple loci and may be particularly useful for analyzing the methylation levels of candidate genes in tumor tissue or cells. It can also be used to detect changes in DNA methylation in other situations, including normal cellular differentiation and aging.

In the MethylCollector MBD Capture Kit, His-tagged recombinant MBD2b protein specifically binds CpG-methylated DNA fragments that have been prepared by enzymatic digestion or sonication. These protein-DNA complexes are captured with nickel-coated magnetic beads and subsequent wash steps are performed to remove fragments with little or no methylation. The methylated DNA is then eluted from the beads in the presence of Proteinase K. Due to the high efficiency of the MethylCollector MBD Capture protocol and the enormous amplification capability and specificity of PCR, analysis of the methylation status of a specific genomic DNA locus can be performed on DNA isolated from less than 170 cells cells (~1 ng DNA).

The MethylCollector MBD Capture Kit is for research use only. Not for use in diagnostic procedures.

MethylCollector MBD Capture Kit

MethylCollector™ MBD Capture Kit Highlights:

  • Detect methylated CpGs from 1 ng – 1 µg of fragmented DNA
  • Finish in less than 3 hours, using magnetic bead-based protocol
  • Check your results using included positive control DNA and qPCR primers
  • Optimize for your regions of interest using either high or low salt binding conditions
  • Use eluted DNA to analyze a specific locus of interest or sequence to analyze whole-genome methylation profiles
 

MethylCollector™ MBD Capture Kit Contents

  • His-MBD2b protein; Store at -80°C
  • High Salt Binding Buffer; Store at -20°C
  • Low Salt Binding Buffer; Store at -20°C
  • Elution Buffer AM1; Store at -20°C
  • Protease Inhibitor Cocktail; Store at -20°C
  • Proteinase K; Store at -20°C
  • Proteinase K Stop Solution; Store at -20°C
  • Human, male genomic DNA, Mse I digested; Store at -20°C
  • GAPDH PCR Primer Mix; Store at -20°C
  • Xist PCR Primer Mix; Store at -20°C
  • NBR2 PCR Primer Mix; Store at -20°C
  • 10X PCR Buffer; Store at -20°C
  • 10X PCR Loading Dye; Store at -20°C
  • Magnetic Nickel Beads; Store at -4°C
  • Glycogen; Store at -20°C
  • Bar Magnet; Store at RT
  • Mini Glue Dots; Store at RT
  • 8-strip PCR tubes and caps; Store at RT
 

MethylCollector™ MBD Capture Kit Data

A.NBR2 high salt
B.Xist low salt
C.GAPDH high salt
D.GAPDH low salt

Figure 1: Real time PCR analysis of control PCR primer sets.
MethylCollector MBD Capture was performed using 100 ng of Mse I digested human, male genomic DNA under both low and high salt conditions. Eluted DNA was purified and analyzed using real time PCR for both methylated and unmethylated promoters. A) Amplification plot using the provided NBR2 PCR primer mix with the unbound and eluted fractions under high salt binding conditions. NBR2 is methylated in the control DNA and shows early amplification of the eluted fractions. B) Amplification plot using the provided Xist PCR primer mix with the unbound and eluted fractions under low salt binding conditions. Xist is methylated in the control DNA and shows early amplification in the eluted fractions. C) Amplification plot using the provided GAPDH PCR primer mix with the unbound and eluted fractions under high salt binding conditions. GAPDH is unmethylated in the control DNA and shows late amplification of the eluted fractions. D) Amplification plot using the provided GAPDH PCR primer mix with the unbound and eluted fractions under low salt binding conditions. GAPDH is unmethylated in the control DNA and shows late amplification of the eluted fractions.


A.NBR2 Enrichment
B.Xist Enrichment
C. GAPDH Enrichment

Figure 2: Percent enrichment of MethylCollector MBD Capture binding reactions.
MethylCollector MBD Capture was performed with 100 ng of Mse I digested human, male genomic DNA using both high and low salt binding conditions. Unbound and eluted DNA was cleaned and analyzed in real time PCR. The amount of DNA recovered in the eluted fraction was divided by the amount of input DNA used in the binding reaction to produce a percent enrichment. A) NBR2 promoter enrichment under high salt conditions. B) Xist promoter enrichment under high salt conditions. C) GAPDH promoter enrichment under both high and low salt conditions. The methylated promoters NBR2 and Xist had greater than 10-fold enrichment of eluted DNA as compared to unbound DNA for the same locus, while the unmethylated GAPDH promoter was found almost exclusively in the unbound fraction.

 

 

MethylCollector™ MBD Capture Kit Documents

 

 

 

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