|
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. 
|
