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. 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.
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 MethylMagnet mCpG DNA Isolation Kit (Catalog # MM101K).
- 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 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 that can be chemically modified to add other reporters or affinity tags, such as biotin, fluorescein or other functional groups.