5-Methylcytosine (5-mC) antibody (mAb)

RRID: AB_2687950
Clone: 33D3
Catalog No: 39649 Format: 50 µg ¥70,000 Buy

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

Applications

Western Blot Validated Methyl-DNA Immunoprecipitation Validated Immunofluorescence Validated Dot Blot Validated Immunohistochemistry Validated Enzyme-linked Immunosorbent Assay Validated Immunocytochemistry Validated

Application Notes


Validated Applications:
MeDIP: 1 µg per IP
IHC (FFPE): 1:1000
WB: 1:250 dilution
ELISA: 1:10,000 dilution

Published Applications:
MeDIP
ICC/IF
IHC
DB
See references for more information. Individual optimization may be required.

Methods for detecting 5-mC in Immunocytochemistry may be found in the following publication: Inoue, A. and Zhang, Y. 2011. Science. Oct 14;334(6053):194. doi: 10.1126/science.1212483. Epub 2011 Sep 22.

Immunogen

Clone 33D3 recognizes the modified base 5-methylcytidine found in plant and vertebrate DNA.

Buffer

Purified IgG in 10 mM phosphate, 0.15 M NaCl and 0.01% thimerosal. Thimerosal is highly toxic.

References

 

5-Methylcytosine antibody (mAb) (Clone 33D3) tested by Methyl-DNA Immunoprecipitation (MeDIP).
Human DNA (500 ng) digested with Mse I was subjected to MeDIP and then analyzed by quantitative real-time PCR with primers specific for genes that are normally methylated (Xist, NYESO and SNRPN) or unmethylated (APC and p48). The MeDIP'd DNA (enriched DNA) was plotted.

5-Methylcytosine (5-mc) antibody (pAb) tested by Immunohistochemistry
Punctate nuclear staining pattern is detected in Formalin-fixed, paraffin-embedded tissue sections from human substantia nigra (midbrain). Top Panel: 5-mC antibody at 1:1000 dilution. Bottom Panel: No primary antibody (2nd step antibody alone)

Background

5-Methylcytosine (5-Methylcytidine) is a modified base that is found in the DNA of plants and vertebrates. 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. The 5-methylcytidine antibody (Clone 33D3) has been developed to discriminate between the modified base and its normal cytosine counterpart, allowing for gene promoter methylation analysis.

qPCR Primers

Active Motif's Human Positive Control Primer Set ZC3H13 (Cat. No. 71009) and Human Negative Control Primer Set 1 (Cat. No. 71001) have been validated for performing qPCR and endpoint PCR when this antibody has been used for ChIP on human samples. For ChIP experiments with this antibody on mouse samples, the Mouse Negative Control Primer Set 1 (Cat. No. 71011) has been validated for both qPCR and endpoint PCR.

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-Methylcytosine (5-mC) 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