5-Hydroxymethylcytosine (5-hmC) antibody (mAb)

RRID: AB_2566808
Clone: 59.1
Catalog No: 39999 Format: 100 µg $460 Buy
Catalog No: 40000 Format: 10 µg $115 Buy
Catalog No: 40900 Format: 50 µg $260 Buy

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Antibody Type:
Protein G Chromatography
Human, Mouse, Not Species Specific


Methyl-DNA Immunoprecipitation Validated Immunofluorescence Validated Dot Blot Validated Immunohistochemistry Validated Immunocytochemistry Validated

Application Notes

Applications Validated by Active Motif:
MeDIP: 1 - 2 µg per IP
MeDIP-chip: 10 µg per IP
DB: 0.2 mg/ml dilution

Published Applications

The following applications have been published using this antibody. Unless noted above, Active Motif may not have validated the antibody for use in these applications:


View publications that use Active Motif products & services here. Enter the product number(s) to see publications & applications that use this antibody.


This 5-Hydroxymethylcytosine antibody was raised against 5-hydroxymethylcytosine conjugated to KLH and recognizes 5-hydroxymethylcytosine.


Purified IgG in PBS, 30% glycerol and 0.035% sodium azide. Sodium azide is highly toxic.


5-Hydroxymethylcytosine (5-hmC) antibody (mAb) - 100 µg (Cat. No. 39999)



hMeDIP-chip performed on human brain DNA using 5-Hydroxymethylcytosine (5-hmC) antibody.
Human brain DNA (2 µg) was immunoprecipitated with 10 µg of 5-Hydroxymethylcytosine antibody. Following hMeDIP, the DNA was amplified, labeled and hybridized to an Affymetrix Human Tiling 2.0R Array. Shown is a region from chromosome 6q containing the ARID1B, ZDHHC14 and SNX9 genes. The results show that 5-hydroxymethylcytosine is enriched primarily in the coding regions of genes, rather than in the gene promoter or regulatory regions.

5-Hydroxymethylcytosine (5-hmC) antibody tested by Methyl DNA immunoprecipitation.
DNA (25 pg) derived from the promoter of the APC gene was spiked into 500 ng of human genomic DNA and subjected to the MeDIP procedure using 2 μg of 5-Hydroxymethylcytosine antibody (5hmC, maroon bars) or 2 μg of control rabbit IgG (IgG, blue bars). Real time quantitative PCR was performed on the immunoprecipitated DNA and results plotted as % of input DNA. The spiked APC DNA contained either no methylation (DNA), 5-methylcytosine methylation (5-mC) or 5-hydroxymethylcytosine methylation (5-hmC).

5-Hydroxymethylcytosine (5-hmC) antibody (mAb) tested by dot blot analysis.
DNA samples were spotted (indicated in ng on the left) on to a positively charged nylon membrane and blotted with 5-Hydroxymethylcytosine antibody at a 0.2 µg/ml dilution.

Lane 1: double-stranded DNA containing 5-hydroxymethylcytosine.
Lane 2: double-stranded DNA containing 5-methylcytosine.
Lane 3: unmethylated double-stranded DNA.
Lane 4: single-stranded DNA containing 5-hydroxymethylcytosine.
Lane 5: single-stranded DNA containing 5-methylcytosine.
Lane 6: unmethylated single-stranded DNA.

Dot blot of 5-Hydroxymethylcytosine (5-hmC) mAb.
Dot blot analysis was used to confirm the specificity of 5-Hydroxymethylcytosine antibody for 5-hydroxymethylcytosine. 10 ng of single-stranded 38 nt DNA oligonucleotides were spotted onto nitrocellulose and probed with the antibody at 0.2µg/mL. Lane 1: oligo containing unmodified cytosine. Lane 2: oligo containing 5-methylcytosine (5-mC). Lane 3: oligo containing 5-hydroxymethylcytosine (5-hmC). Lane 4: oligo containing 5-formylcytosine (5-fC). Lane 5: oligo containing 5-carboxylcytosine (5-caC).


In addition to this monoclonal, Active Motif offers two polyclonal antibodies that recognize 5-hydroxymethylcytosine, a whole serum version (39769) and a purified IgG version (39791).All are validated for use in methyl DNA immunoprecipitation (MeDIP). For customers that require the ability to quantitate the amount of IgG in the MeDIP reaction, we recommend either this monoclonal or the purified IgG polyclonal (39791). The whole serum version (39769) is very high titre and should be used carefully (0.1 - 0.5 µl per IP) as not to generate high non-specific background. The whole serum version (39769) has been used successfully in immunofluorescence (IF, Ito et al, 2010), and the purified IgG version (39791) is likely to work in this application as well.

DNA methylation is an epigenetic event in which DNA methyltransferases (DNMTs) catalyze the reaction of a methyl group to the fifth carbon of cytosine in a CpG dinucleotide. This modification helps to control gene expression and is also involved in genomic imprinting, while aberrant DNA methylation is often associated with disease. 5-methylcytosine is a modified base that is found in the DNA of plants and vertebrates.
A second type of DNA methylation exists, 5-hydroxymethylcytosine (5-hydroxy methyl cytosine, 5-hmC). This results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of cytosine oxygenases. This antibody was developed specifically to distinguish 5-hydroxymethylcytosine from 5-methylcytosine as conventional methods (enrichment by antibody or methyl DNA binding protein, enzymatic digestion and bisulfite sequencing) cannot do so.
It is possible that 5-hydroxymethylcytosine (5-hmC) represents a pathway to demethylate DNA, as 5-hydroxymethylcytosine is repaired as mismatched DNA and replaced with unmethylated cytosine.


Some products may be shipped at room temperature. This will not affect their stability or performance. Avoid repeated freeze/thaw cycles by aliquoting items into single-use fractions for storage at -20°C for up to 2 years. Keep all reagents on ice when not in storage.


This product is guaranteed for 12 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.

Application Key

  • ChIP = Chromatin Immunoprecipitation;
  • ChIP = ChIP Sequencing;
  • CUT&RUN = Cleavage Under Targets and Release Using Nuclease;
  • CUT&Tag = Cleavage Under Targets and Tagmentation;
  • DB = Dot Blot;
  • ELISA = Enzyme-linked Immunosorbent Assay;
  • EMSA = Electrophoretic Mobility Shift Assay
  • FC = Flow Cytometry;
  • ICC = Immunocytochemistry;
  • IF = Immunofluorescence;
  • IHC = Immunohistochemistry;
  • IP = Immunoprecipitation;
  • MeDIP = Methyl-DNA Immunoprecipitation;
  • NEU = Neutralizing;
  • WB = Western Blotting