5-Hydroxymethylcytosine (5-hmC) antibody (rAb)

Clone: RM236
Catalog No: 91309 Format: 50 µg ¥70,000 Buy

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Antibody Type:
Recombinant
Isotype:
IgG
Purification:
Protein A Chromatography
Host:
Rabbit
Reactivity:
Human, Not Species Specific

Applications

Immunofluorescence Validated Dot Blot Validated Immunohistochemistry Validated

Application Notes


Validated Applications:
DB: 0.2 - 1 µg/ml
IHC: 0.1 - 1 µg/ml
ICC: 0.5 - 2 µg/ml
MeDIP: 0.2 - 2 µg/ml

Immunogen

BSA-conjugated 5-hydroxymethylcytosine.

Buffer

PBS with 50% glycerol, 1% BSA, and 0.09% sodium azide.

References

J. Reichmann et al. "Dual spindle formation in zygotes keeps parental genomes apart in early mammalian embryos." bioRxiv (2017) 10.1101/198275.
Siref, A.B. et al. "Diagnostic utility of dual 5-hmC/Melan-A immunohistochemistry in differentiating nodal nevus from metastatic melanoma: An effective first-line test for the work-up of sentinel lymph node specimen." J. Cutan Pathol. (2019) 10.1111/cup.13412.

 

Dot blot of 5-Hydroxymethylcytosine (5-hmC) antibody (rAb).
Membrane was pre-spotted with 50, 5, and 0.5 ng/dot of double stranded 5-Hydroxymethylcytosine (5-hmC) DNA, 5-Methylcytosine (5-mC) DNA, and unmethylated DNA. The pre-spotted membrane was then blotted with antibody at 0.2 µg/ml.

MeDIP of 5-Hydroxymethylcytosine (5-hmC) antibody (rAb).
MeDIP was performed using 5-Hydroxymethylcytosine antibody (Clone RM236) at a 10:1 DNA:Ab ratio. 1 ng of unmethylated, 5-Methylcytosine (5-mC) or 5-Hydroxymethylcytosine (5-hmC) DNA standard (897 bp) was spiked in 1 µg of genomic DNA isolated from HeLa cells as the control. Realtime PCR was then performed to determine the capture of DNA standard as in % of input. 

Immunofluoresence staining of HeLa cells using 5-Hydroxymethylcytosine antibody (Clone RM236) at 0.5 µg/mL (red). Actin filaments are labeled with fluorescein phalloidin (green). HeLa cells were fixed with 4% paraformaldehyde and permeabilized with methanol (−20°C) before treatment with 2 N HCl for 30 min at 37°C to denature DNA.

Immunohistochemical staining of formalin fixed, paraffin embedded human brain tissue sections, using 5-Hydroxymethylcytosine antibody (Clone RM236) at 1 µg/ml.

Background

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;
  • 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