YOUR CART
Active Motif's MethylCollector™ Kit provides an efficient method* for isolating CpG-methylated DNA from limited amounts of cell or tissue samples. MethylCollector has many powerful applications such as screening the methylation status of multiple loci and it is 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.
*Technology covered under U.S. Patent No. 7,425,415.
Advantages
- Fast and easy protocol is complete in less than 4 hours
- Flexible – enables detection from 5 ng to 1 µg of DNA
- Suitable for use with DNA fragmented by sonication or enzymatic digestion
- Positive control DNA and PCR primers ensure success
In MethylCollector, 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 with a stringent high-salt buffer to remove fragments with little or no methylation. The methylated DNA is then eluted from the beads.
Figure 1: Flow chart of the MethylCollector process.
Due to the high efficiency of MethylCollector and the enormous amplification and specificity of PCR, analysis of the methylation status of a specific genomic DNA locus can be performed on DNA isolated from less than 800 cells (~5 ng DNA).
Figure 2: Fully Methylated Jurkat DNA used as a control for MethylCollector.
The MethylCollector Kit was used to assay 100 ng genomic DNA (lanes 2 & 4) and Fully Methylated Jurkat DNA (lanes 3 & 5) samples with (lanes 4 & 5) or without (lanes 2 & 3) inclusion of 1 µg of the kit’s His-tagged MBD2b protein. These samples, the input genomic DNA (lane 6) and the input Fully Methylated Jurkat DNA (lane 7) were then PCR amplified with the BRCA1 primers. Lane 8 is a water-only PCR control. A positive result is observed only with Fully Methylated Jurkat DNA captured by MethylCollector’s His-tagged MBD2b (lane 5), demonstrating that MethylCollector specifically isolates methylated DNA and that Fully Methylated Jurkat DNA is a useful control in methylation-specific experiments.
