The MethylCap assay is based on the affinity purification of methylated DNA using a MBD (Methylbinding domain) protein . This procedure has first been optimized on the SX-8G IP-Star Automated System ([page product SX-8G IP-Star].
Afterwards, this method has been adapted to a manual protocol, which has been inspired by the same reproducibility standards provided by the automated MethylCap assay.
Methylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of promoter CpGs being hypomethylated (Kangaspeska, 2008).
The Diagenode H6-GST-MBD fusion protein of the MethylCap kit has been extensively validated. It consists of the methyl binding domain (MBD) of human MeCP2, as a C-terminal fusion with Glutathione-S-transferase (GST) containing an N-terminal His6-tag. The H6-GST-MBD fusion protein can be used to specifically isolate DNA containing methylated CpGs.