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

RRID: AB_2566808
Clone: 59.1
Catalog No: 39999 Format: 100 µg ¥70,000 Buy
Catalog No: 40000 Format: 10 µg ¥20,000 Buy
Catalog No: 40900 Format: 50 µg ¥40,000 Buy

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Antibody Type:
Monoclonal
Isotype:
IgG2a
Purification:
Protein G Chromatography
Host:
Mouse
Reactivity:
Human, Mouse, Not Species Specific

Applications

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

Application Notes


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

Published Applications:
MeDIP
ICC/IF
IHC (FFPE)

See references for more information. Individual optimization may be required.

Immunogen

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

Buffer

Purified IgG in 70 mM Tris (pH 8), 105 mM NaCl, 31 mM glycine, 0.07 mM EDTA, 30% glycerol and 0.035% sodium azide. Sodium azide is highly toxic.

References

 

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, 5-hydroxymethylcytidine) 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-Hydroxymethylcytidine 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-Hydroxymethylcytidine 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-hydroxymethylcytidine. 10 ng of single-stranded 38 nt DNA oligonucleotides were spotted onto nitrocellulose and probed with the antibody at 0.2mg/mL. Lane 1: oligo containing unmodified cytidine. Lane 2: oligo containing 5-methylcytidine (5-mC). Lane 3: oligo containing 5-hydroxymethylcytidine (5-hmC). Lane 4: oligo containing 5-formylcytidine (5-fC). Lane 5: oligo containing 5-carboxylcytidine (5-caC).

Background

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 methylcytosine, 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.

Bridging Antibody to Improve ChIP, MeDIP and IP

Some isotypes of mouse IgG antibodies do not bind well to protein G-conjugated agarose/magnetic beads, which are commonly used in ChIP, MeDIP and IP experiments. Capture efficiency can be improved by including an anti-mouse IgG bridging antibody because it binds with a strong affinity to both the protein G beads and the mouse IgG antibody. By increasing the affinity of the mouse primary antibody for the protein G bead, the Bridging Antibody for Mouse IgG can help improve the results of all ChIP, MeDIP and IP experiments that use 5-Hydroxymethylcytosine (5-hmC) antibody (mAb).

Storage

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.

Guarantee

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;
  • 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;
  • TR-FRET = Time-Resolved Fluorescence Resonance Energy Transfer;
  • WB = Western Blotting