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Catalog #

Description

Size

Price

ICM1

ICM for DNMT1

50 Assays

See Price List

ICM1B

ICM for DNMT1b

50 Assays

See Price List

ICM3A

ICM for DNMT3a

50 Assays

See Price List

ICM3B

ICM for DNMT3b

50 Assays

See Price List

ICM2

ICM for DNMT2

50 Assays

See Price List

ICM3L

ICM for DNMT3L

50 Assays

See Price List

The ICM Kits are based on the finding that DNMT becomes irreversibly (covalently) bound to genomic DNA that is labeled with aza-deoxycytidine. This allows researchers to measure DNMT binding in vivo using antibodies (to specific isoforms of DNMT) to probe the DNA peak fractions of a CsCl gradient. Essentially, the ICM is a way to detect real time methylation activity on specific gene regions with specific DNMT isoforms. The method is based on the published paper by Liu et al, (2003) and is described in more detail in the PDF file:  ICM KIT SUMMARY.

 Each of our kits is opitimized for a given DNMT-x isoform (1, 2,3 etc). See price list for all kit listings. 

An overview of the ICM Technology is illustrated below:

Additional comments: 
The key to the analysis is to separate DNA from free protein in a single step using CsCl density separation. One then uses Western slot or dot blots of the DNA peak to identify the presence of a given DNMT isoform. As shown above, cells labeled with the hypomethylating drug 5-aza-2’-deoxycytidine, have DNMT-1 in the DNA fraction while control cells (no drug) have no detectable DNMT-1. Moreover, probing with a non-specific antibody (topoisomerase), fails to detect any signal in the DNA fractions either in the presence or absence of aza-deoxycytidine. Note also that the amount of signal is directly proportional to the amount of DNMT-1 active on the genome at the time of lysis by sarkosyl.

To demonstrate the quantitative aspects of the ICM Kit, the following experiment was done. HeLa cells were labeled for 1 hour with increasing amounts of aza-dC and DNA analyzed by ICM. The DNA fractions were pooled and its concentration accurately measured by fluorometry. Either 1,5 or 10 ug of DNA were slot blotted and probed with anti-DNMT1 antibody. The data show increasing amounts of DNMT-1 on the genome with increasing aza-dC treatment. If one is interested in a quantitative comparison of different isoforms, the ICM Kit should work extremely well using this quantitative adaptation.

What is included in the ICM Kit:

  • Technical Instructions
  • CsCl Stock Solutions
  • Antibody (Specify DNMT isoform, see Price list).
  • Sarkosyl Lysis solution
  • Hypomethylating drug

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