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Antibodies

5-Hydroxymethylcytosine (5-hmC) antibody (pAb)

Catalog No: 39769 Format: 100 µl $410 Buy Now
Catalog No: 39770 Format: 10 µl $95 Buy Now

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Antibody Type:Polyclonal
Isotype:Serum
Purification:None
Host:Rabbit
Reactivity:Human, Mouse, Other (Wide Range)

Applications

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

for 5-Hydroxymethylcytosine (5-hmC) antibody (pAb)Application Notes


Validated Applications:
MeDIP: 0.1 - 0.5 µl per IP
DB: 1:10,000 dilution

Published Applications:
MeDIP
MeDIP-Seq
ICC/IF
IHC
FC
DB

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

for 5-Hydroxymethylcytosine (5-hmC) antibody (pAb)Immunogen

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

for 5-Hydroxymethylcytosine (5-hmC) antibody (pAb)Buffer

Rabbit serum containing 30% glycerol and 0.035% sodium azide. Sodium azide is highly toxic.

for 5-Hydroxymethylcytosine (5-hmC) antibody (pAb)References

 
5-Hydroxymethylcytosine (5-hmC) antibody (pAb) tested by MeDIP analysis.

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 1 μl of 5-Hydroxymethylcytidine antibody (5hmC, maroon bars) or 1 μl 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 (pAb) tested by dot blot analysis.

5-Hydroxymethylcytosine (5-hmC, 5-hydroxymethylcytidine) antibody tested by dot blot analysis.
DNA samples (10 ng or 50 ng as indicated) were spotted onto positively charged nylon membrane and blotted with 5-Hydroxymethylcytidine antibody at a dilution of 1:10,000.
Top Panel: Double stranded DNA.
Bottom Panel: Single stranded DNA.
Lane 1: Unmethylated DNA.
Lane 2: DNA containing 5-methylcytosine.
Lane 3: DNA containing 5-hydroxymethylcytosine.

5-Hydroxymethylcytosine (5-hmC) antibody (pAb) tested by dot blot analysis.

5-Hydroxymethylcytosine (5-hmC, 5-hydroxymethylcytidine) antibody tested by dot blot analysis.
DNA samples were spotted onto positively charged nylon membrane and blotted with antibodies as indicated.
Panel A: 5-Hydroxymethylcytidine antibody recognizing 5-hydroxymethylcytosine (1:10,000 dilution).
Panel B: 5-Methylcytidine antibody (1:1,000 dilution).

Lane 1: DNA derived from mouse embryonic stem cells (150 ng).
Lane 2: DNA derived from mouse spleen (600 ng).
Lane 3: 27 base oligonucleotide containing 5-hydroxymethylcytosine (1.2 ng).
Lane 4: 33 base oligonucleotide containing 5-methylcytosine (2000 ng).

for 5-Hydroxymethylcytosine (5-hmC) antibody (pAb)Background

Active Motif offers two polyclonal antibodies that recognize 5-hydroxymethylcytosine, a whole serum version (39769) and a purified IgG version (39791). Both are validated for use in methyl DNA immunoprecipitation (MeDIP). For customers who must quantitate the amount of IgG in the MeDIP reaction, the purified IgG version (39791) is recommended. The whole serum version (39769) is very high titre, so should be used carefully (0.1 - 0.5 µl per IP) to prevent non-specific background. The whole serum version (39769) has been used successfully in immunofluorescence (IF, Ito et al, 2010);the purified IgG version (39791) is likely to work in IF 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.

Compatible Secondary Antibodies

Active Motif has developed a variety of high-quality secondary antibodies for most applications, with anti-rabbit and anti-mouse conjugates to a broad line of high-quality fluorescent molecules, as well as horse radish peroxidase (HRP). Visit our Secondary Antibody Conjugates page to find the perfect secondaries for immunofluorescence experiments with 5-Hydroxymethylcytosine (5-hmC) antibody (pAb).

for 5-Hydroxymethylcytosine (5-hmC) antibody (pAb)Storage

Some products may be shipped at room temperature. This will not affect their stability or performance. Upon receipt, unconjugated antibodies may be stored at -20°C for up to 2 years. Fluorophore- & enzyme-conjugated antibodies should be stored at 4°C. Fluorophore-conjugated antibodies should be protected from light. Keep reagents on ice when not in storage; to avoid repeated freeze/thaw cycles, we recommend aliquoting items that will be stored frozen into single-use fractions prior to freezing.

for 5-Hydroxymethylcytosine (5-hmC) antibody (pAb)Guarantee

This product is guaranteed for 6 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
 

Technical Data Sheet

5-Hydroxymethylcytosine (5-hmC) antibody (pAb)
 

Data Thumbnails

5-Hydroxymethylcytosine (5-hmC) antibody (pAb) tested by MeDIP analysis.

MeDIP of 5-Hydroxymethylcytosine (5-hmC) pAb.
(Click image to enlarge and see details.)

5-Hydroxymethylcytosine (5-hmC) antibody (pAb) tested by dot blot analysis.

Dot blot of 5-Hydroxymethylcytosine (5-hmC) pAb.
(Click image to enlarge and see details.)

5-Hydroxymethylcytosine (5-hmC) antibody (pAb) tested by dot blot analysis.

Dot blot of 5-Hydroxymethylcytosine (5-hmC) pAb.
(Click image to enlarge and see details.)