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Applications: hMeDIP (2.5 µg/IP)
DB (2 µg /ml)
ELISA (1:500)
IF
Assay Conditions: An hydroxymethylated DNA IP (hMeDIP) was performed using the mouse monoclonal antibody directed against 5-hydroxymethylcytosine (#25202). The IgG isotype antibodies from mouse were used as negative control. The DNA was prepared with the GenDNA module of the hMeDIP kit and sonicated 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 immunoprecipitated material was analyzed 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).Determination of the 5-hmC mouse monoclonal antibody titer: To determine the titer, an ELISA was performed using a serial dilution of the mouse monoclonal antibody directed against 5-hmC (#25202) 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.Dotblot analysis of the 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 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.
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: 20 µg/20 µl
Cross Reactivity: Specific for 5-hmc. Does not react with 5-mc or unmodified cytosine.
Description: Monoclonal antibody raised in mouse against 5-hydroxymethylcytosine conjugated to BSA
Format: Aqueous buffer solution
Formulation: PBS (ph 7.4) containing 0.05% sodium azide
Immunogen: nucleotide
Purification: Protein A/G 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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