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

Application Notes

Applications Validated by Active Motif:
MeDIP: 0.1 - 0.5 µl per IP
DB: 1:10,000 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:

MeDIP, MeDIP-Seq
ICC/IF
IHC(P), IHC(F)
FC
DB
ELISA

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

Immunogen

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

Buffer

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

References

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.

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;
• 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