Anti–5–hmC polyclonal antibody (rabbit)

Catalog #: 25203
Size: 50 µg
Host Species: Rabbit
Clonality: polyclonal
5–hydroxymethylcytosine (5–hmC) results from the enzymatic conversion of 5–methylcytosine into 5–hydroxymethylcytosine by the TET family of iron–dependent oxygenases. 5–hmC bases were recently discovered in mammalian DNA, in Purkinje neurons, in granule cells and embryonic stem cells where theyare present at high levels (up to 0.6% of total nucleotides in Purkinje cells). Recent reports indicate that 5–hmC is abundant in brain tissue, especially in areas that are associated with higher cognitive functions. Preliminary results indicate that 5–hmC may have important roles distinct from 5–mC. Although its precise role has still to be shown, early evidence suggests 5–hydroxymethylcytosine may represent a new and unique pathway to demethylate DNA involving a repair mechanism converting 5–hmC to cytosine. Due to the structural similarity between 5–mC and 5–hmC, these bases are experimentally almost indistinguishable. The most commonly used methodologies (e.g. enzymatic approaches, bisulfite sequencing) do not distinguish 5mc from 5–hmC. The development of specific antibodies appears to be the most powerful way to distinguish and specifically enrich for 5–mC and 5–hmC sequences.
Polyclonal antibody raised in rabbit against 5–hydroxymethylcytosine conjugated to KLH.
Assay Conditions:

hMeDIP results obtained with the antibody directed against 5-hmC
hMeDIP (hydroxymethylated DNA IP) was performed using the antibody against 5-hydroxymethylcytosine (Cat. No. 25203). DNA from mouse ES cells was prepared with the GenDNA module of the hMeDIP kit and sonicated to obtain DNA fragments of 300-500 bp. One μg of sheared DNA was spiked with unmethylated (C) methylated (mC), and hydroxymethylated (hmC) controls. hMeDIP was performed with 3.5 μg of the rabbit 5-hmC antibody and the immunoprecipitated DNA was analysed by qPCR using primers specific for the 3 different control sequences. Figure 1 shows that the rabbit polyclonal antibody against 5-hmC is highly specific for the 5-hmC base modification (no IP with non-methylated or methylated C control fragments).

Determination of the antibody titer
To determine the titer, an ELISA was performed using a serial dilution of the antibody directed against 5-hmC (cat. No. 25203), crude serum and flow through, in antigen coated wells. The antigen used was the 5-hmC base coupled to BSA. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:2,800.

Dot blot analysis using the antibody directed against 5-hmC
To demonstrate the specificity of the antibody against 5-hmC (cat. No. 25203), a Dot blot analysis was performed using hmC, mC and C controls. One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane (Amersham Hybond-N+). The antibody was used at a dilution of 1:1,000. Figure 3 shows a high specificity of the antibody for the hydroxymethylated control.

50 µg
PBS containing 0.05% azide and 0.05% ProClin 300.
Species Reactivity:
Human, mouse, broad range
Affinity purified
Aqueous buffer solution
Storage / Stability:

Store at –80°C for up to 2 years. Centrifuge after first thaw to maximize product recovery. Aliquot to avoid repeated freeze/thaw cycles. Aliquots may be stored at –20°C for at least one month.

hMeDIP (3.5 µg/IP)
ELISA (1:100 – 1:500)
DB (1:1000)
The optimal antibody concentration should be determined by the end–user.
Avoid freeze/thaw cycles
Scientific Category:
Product Type:
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