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Applications: hMeDIP (3.5 µg/IP)
ELISA (1:100 - 1:500)
DB (1:1000)
Assay Conditions: hMeDIP results obtained with the antibody directed against 5-hmC
hMeDIP (hydroxymethylated DNA IP) was performed using the antibody against 5-hydroxymethylcytosine (#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.
Background: 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.
Concentration: 50 µg
Description: Polyclonal antibody raised in rabbit against 5-hydroxymethylcytosine conjugated to KLH.
Format: Aqueous buffer solution
Formulation: PBS containing 0.05% azide and 0.05% ProClin 300.
Immunogen: nucleotide
Purification: Affinity purified
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.
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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