5-hmC | 5-hydroxymethylcytosine (monoclonal)
AS16 3164 | clonality: monoclonal | host: mouse | reactivity:human, mouse, other species
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|Recommended dilution||2 ĩg/ml (Dot), 1 : 500 (ELISA), 2.5 ĩg/IP (hMeDIP)|
|Expected | apparent MW|
|Predicted reactivity||Mouse, broad species range|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
|Selected references||To be added when available, antibody released in February 2016.|
An hydroxymethylated DNA IP (hMeDIP) was performed using the mouse monoclonal antibody directed against 5-hydroxymethylcytosine. The IgG isotype antibodies from mouse was used as negative control. The DNA was prepared with the GenDNA module of the hMeDIP kit and sonicated with our Bioruptor ® (UCD-200/300 series) to have DNA fragments of 300-500 bp. 1 μg of human Hela cells DNA were spiked with non-methylated, methylated, and hydroxymethylated PCR 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 mouse monoclonal 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 mouse monoclonal antibody directed against 5-hmC in antigen coated wells. The antigen used was KHL coupled to 5-hmC base. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:40 000.
Dot blot: using anty-5-hmC mouse monoclonal antibody with the C, mC and hmC PCR controls 200 to 2 ng (equivalent of 10 to 0.1 pmol of C-bases) of the hmC (1), mC (2) and C (3) PCR controls from the “5-hmC, 5-mC & cytosine DNA were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 2 μg/ml of the mouse 5-hydroxymethylcytosine monoclonal antibody (dilution 1:500). The membranes were exposed for 30 seconds.
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