5-hmC | 5-hydroxymethylcytosine (polyclonal)
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|Recommended dilution||1: 200 (Dot), 1: 500 (ELISA), 2.5 µl/IP (hMeDIP)|
|Expected | apparent MW|
|Predicted reactivity||mouse, broad species range|
|Not reactive in||No confirmed exceptions from predicted reactivity known at the moment|
|Additional information||To be added when available|
|Selected references||To be added when available, antibody released in February 2016.|
An hydroxymethylated DNA IP (hMeDIP) was performed using the rabbit polyclonal antibody directed against 5-hydroxymethylcytosine. The IgG isotype antibodies from rabbit were used as negative control. The DNA was prepared with the GenDNA module of the hMeDIP kit and sonicated with our Bioruptor® to have DNA fragments of 300-500 bp. 1 μg of human Hela cells DNA were spiked with non-methylated, methylated, and hydroxymethylated fragments. The IP’d material has been analysed by qPCR using the primer pair specific for the 3 different control sequences. The obtained results show that the rabbit polyclonal for 5-hmC is highly specific for this base modification (no IP with non-methylated or methylated C bases containing fragments).
ELISA: to determine the titer, an ELISA was performed using a serial dilution of the rabbit polyclonal antibody directed against 5-hmC in antigen coated wells. The antigen used was BSA coupled to the 5-hmC base. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1: 3 500.