5-hmC Antibody (C.15220001)

Methylated DNA Immunoprecipitation: 5-hmC Antibody (C.15220001) [NBP2-59178]

Methylated DNA Immunoprecipitation: 5-hmC Antibody (C.15220001) [NBP2-59178] - Hydroxymethylated DNA IP (hMeDIP) assays were performed using the antibody against 5-hydroxymethylcytosine, 5-hmC, 5-mC and cytosine DNA standards and Rat IgG. DNA fragments are 300-500 bp. 1 ug of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP'd material has been analyzed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control, with Rat IgG as negative control (0.06%, almost no recovery), with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control, with Rat IgG as negative control (0.03%, almost no recovery), with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control, with Rat IgG as negative control (0.04%, almost no recovery), with 5-hmC (97.60% recovery, almost full recovery).

ELISA: 5-hmC Antibody (C.15220001) [NBP2-59178] - To determine the titer, an ELISA was performed using a serial dilution of the antibody directed against 5-hmC in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.

Dot Blot: 5-hmC Antibody (C.15220001) [NBP2-59178] - (A): Approximately 200 ng, equivalent 10 pmol of C-bases, of the hmC (1), mC (2) and C (3) PCR controls were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hmC Antibody (C.15220001) followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed for 30 seconds.