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MethylMagnet® Protein

Cat# MM101     Methyl Magnet® GST-MBD Protein               100 μg

Cat# MM102     MethylMagnet® GST-MBDX Protein             100 μg

For pricing and to place an order, please contact info@ribomed.com

Overview

MethylMagnet proteins are versatile tools for the study of CpG methylation in DNA.  These proteins contain the methyl binding domain (MBD) of the mouse MBD2 protein fused to the glutathione-S-transferase protein (GST) from S japonicum. The two domains are separated by a linker containing a thrombin cleavage site (Figure 1). The MBD from the MBD2b protein was chosen because MBD2b has the highest affinity among the known methyl CpG binding proteins for Me-CpG sites and the lowest cross reactivity with unmethylated CpGs. It has between a 25 to 100 fold higher affinity for Me-CpG sites than does MeCP2 and a 9.7 to 43 fold higher preference for methylated DNA than does MeCP2. Additionally, there are no sequence context effects on MBD2 CpG recognition, as there are for MeCP2, which requires a run of 4 A-Ts near a CpG site, therefore a greater number of mCpG sites should be recognized by MethylMagnet.

            Figure 1

 

In addition to its high specificity for methylated DNA, MethylMagnet proteins have several unique features that make them useful tools in the study of DNA methylation.

  • The GST allows the fusion protein or its complexes with methylated DNA to be isolated on Glutathione agarose or magnetic beads and eluted intact with glutathione, as with the MethylMeter™ mCpG DNA Isolation Kit.
  • The GST group further allows the eluted protein-DNA complexes to be immobilized to beads or microtiter plates with GST antibodies.
  • Alternatively, the GST group can be used for visualization of the DNA-protein complexes by using labeled GST antibodies or GST antibodies and labeled secondary antibodies, or other detection methods for GST.
  • The thrombin cleavage site (Figure 1A) allows the MBD to be separated from the GST, if desired, although the fusion protein is fully functional for binding to methylated DNA.
  • The GST group contains surface cysteine groups (Figure 1B) that can be chemically modified to add other reporters or affinity tags, such as biotin (link Fig 4), fluorescein or other functional groups.

 

How it Works

MethylMagnet has high affinity for fully methylated DNA sites

 

The MethylMagnet protein has high affinity and specificity for binding to DNA containing 5-Methyl-CpG groups. The protein has much higher affinity for DNA methylated on both strands, versus hemi-methylated or unmethylated DNA. Purified GST-MBD2 was incubated with a 550 bp p16 amplicon that was either fully methylated or unmethylated or with no DNA (Figure 2). The biotinylated DNA target was immobilized to magnetic beads. The GST-MBD2 protein was incubated with the immobilized DNA for 1 hour. Free GST-MBD2 was removed with 3 washes. Bound GST-MBD2 was detected with an anti-GST antibody-horseradish peroxidase conjugate. The HRP reaction was performed for 5 min. The filled and unfilled bars represent duplicate measurements (Figure 3)

Figure 2                                                                                      Figure 3

 

                                   

 

MethylMagnet can be modified with reporter groups and still bind mCpG DNA

 

Both the unmodified GST-MBD protein and one in which biotin had been added through the thiol groups on the GST domain were tested for specific binding to methylated DNA. The Biotin-GST-MBD proteins were determined to have an average of 2 biotins per molecule. Two genomic DNA samples (100 ng) containing methylated (M) or unmethylated (U) p16 islands were cleaved with restriction endonuclease Mse I and incubated for 1 hour with glutathione magnetic beads containing GST-MBD or Biotin-GST-MBD protein. The fractionation of methylated from unmethylated DNAs was performed by pelleting the beads, removing the supernatant and eluting the bead-bound DNA with glutathione buffer. The presence of p16 island sequence in each fraction was measured by PCR.

Figure 4

Lane 1: Eluted M DNA from GST-MBD beads

Lane 2: Eluted U DNA from GST-MBD beads

Lane 3: Eluted M DNA from Biotin-GST-MBD beads

Lane 4: Eluted U DNA from Biotin-GST-MBD beads