5-hmC | 5-hydroxymethylcytosine (polyclonal)

350 €
AS16 3166 clonality: polyclonal  |  host: rabbit  |  reactivity:human, mouse, other species


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Item No:
AS16 3166

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product information
Background 5-hmC | 5-hydroxymethylcytosine is a recently discovered DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. It may have an important roles distinct from that of 5-methylcytosine (5-mC). 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells.
Immunogen BSA-conjugated molecule: 5-hydroxymethylcytosine (5-hmC)
Host Rabbit
Clonality Polyclonal
Quantity 100 ĩl
Storage Store at -20°C; for long term storage store at -80°C; make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tubes.
Tested applications Dot blot (Dot), ELISA (ELISA), hMeDIP (hydroxymethylated DNA immunoprecipitation)
Related products AS16 3164 | anti-5-hmC | 5-hydroxymethylcytosine (monoclonal), mouse antibodies

collection of antibodies to epigenetics
Additional information This serum contains 0.05 % sodium azide.
application information
Recommended dilution 1: 200 (Dot), 1: 500 (ELISA), 2.5 µl/IP (hMeDIP)
Expected | apparent MW
Confirmed reactivity


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.

application example

hMeDIP using polyclonal antibody

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 with polyclonal antibody anti-5-hmC

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.

Dot blot using polyclonal anti 5-hmC antibodies

Dot blot analysis of the 5-hmC rabbit polyclonal antibody with the C, mC and hmC PCR controls 100 to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the hmC, mC and C PCR controls from the “5-hmC, 5-mC & cytosine DNA Standard Pack” were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with the rabbit 5-hydroxymethylcytosine polyclonal antibody (dilution 1:200). The membranes were exposed for 30 seconds.