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

Clone: A1
Catalog No: 61479 Format: 100 µg $435 Buy
Catalog No: 61480 Format: 10 µg $105 Buy

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Antibody Type:
Monoclonal
Isotype:
IgG1
Purification:
Protein A Chromatography
Host:
Mouse
Reactivity:
Human, Not Species Specific

Applications

Methyl-DNA Immunoprecipitation Validated Immunofluorescence Validated Dot Blot Validated

Application Notes


Validated Applications:
MeDIP: 1 - 2 µg per IP
MeDIP-Seq: 1 - 2 µg each
DB: 0.5 - 2 µg/ml dilution

In MeDIP, the DNA must be denatured when using this antibody.

Published Applications:
IF
Individual optimization may be required.

Immunogen

This 5-Methylcytosine (5-mC) antibody was raised against 5-Methyl-cytidine conjugated to KLH and recognizes 5-Methylcytosine.

Buffer

Purified IgG in PBS with 30% glycerol and 0.035% sodium azide. Sodium azide is highly toxic.

Also available as an AbFlex™ engineered recombinant antibody.  For details on the corresponding AbFlex Recombinant Antibody, see Catalog No. 91187.

References

 
5-Methylcytosine (5-mC) antibody (mAb) tested by MeDIP-Seq analysis.

Next-Gen sequencing data generated using 5-Methylcytosine (5-mC) antibody (mAb) correlates well with CpG density.
DNA was enriched from 1 ug of denatured, adaptor ligated human PC9 cell DNA using 2 ul of the 5-Methylcytosine (5-mC) antibody (mAb). MeDIP DNA was sequenced using the Illumina platform to generate 26 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows that the enriched regions (purple peaks) correlate well with CpG density (green overlay).

5-Methylcytosine (5-mC) antibody (mAb) tested by MeDIP analysis.

5-Methylcytosine (5-mC) antibody (mAb) tested by Methyl-DNA Immunoprecipitation (MeDIP).
Human DNA (500 ng) digested with MseI was subjected to MeDIP using 2 µg of the 5-Methylcytosine antibody and then analyzed by quantitative real-time PCR with primers specific for ZC3H13 that is normally methylated or Untr1 that is normally unmethylated. The MeDIP'd DNA (enriched DNA) was plotted.

5-Methylcytosine (5-mC) antibody (mAb) tested by dot blot analysis.

5-Methylcytosine (5-mC) antibody (mAb) tested by dot blot analysis.
DNA from the Methylated DNA Standard Kit (Catalog No. 55008) were spotted (5 ng per spot) on to a positively charged nylon membrane and blotted with 5-Methylcytosine antibody (2 µg/ml dilution).

Lane 1: single-stranded unmethylated DNA.
Lane 2: single-stranded DNA containing 5-methylcytosine.
Lane 3: single-stranded DNA containing 5-hydroxymethylcytosine.
Lane 4: double-stranded unmethylated DNA.
Lane 5: double-stranded DNA containing 5-methylcytosine.
Lane 6: double-stranded DNA 5-hydroxymethylcytosine.

Background

5-Methylcytosine (5-mC) 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 A1) has been developed to discriminate between the modified base and its normal cytosine counterpart, allowing for gene promoter methylation analysis.

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).

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

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.

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