Diagenode

5-methylcytosine (5-mC) monoclonal antibody for ICC/IF - Classic

RFX5-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15200003
50 μg/21 μl
$295.00
  Bulk order

Monoclonal antibody raised in mouse against 5-mC (5-methylcytosine) conjugated to BSA. 

Lot001
Concentration2.4 µg/µl
Species reactivityHuman, mouse, other (wide range)
TypeMonoclonal
PurityProtein A purified
HostMouse
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
MeDIP * 1-2 μg per IP
Dot Blotting 1:600 Fig 1
Immunofluorescence 1:500 Fig 2
FISH 1:200 - 1:1,000 Fig 3, 4
* Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 μg per IP.
  • Validation Data

    Dot Blot

    Figure 1. Dot blot analysis using the Diagenode monoclonal antibody directed against 5-mC
    To demonstrate the specificity of the Diagenode antibody against 5-mC (Cat. No. C15200003), a Dot blot analysis was performed using the hmC, mC and C controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat. No. AF-101-0002). One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane (Amersham Hybond-N+). The antibody was used at a dilution of 1:600. Figure 1 shows a high specificity of the antibody for the methylated control.

    Immunofluorescence

    Immunofluorescence

    Immunofluorescence

    Figure 2. Immunofluorescence results obtained with the Diagenode monoclonal antibody directed against 5-mC
    Human osteosarcoma (U2OS) cells were stained with the Diagenode monoclonal antibody against 5-mC (Cat. No. C15200003). Cells were fixed with 2.5% PFA in PBS for 30’, permeabilised with 0.5% Triton X-100 for 1 hour and treated with 2N HCl for 1 hour B. followed by 2 x 5 minutes with 0.1 M borate buffer to depurinate the DNA. After blocking with PBS containing 0.1% Triton X-100 and 1% BSA, the cells were immunofluorescently labelled with the 5-mC antibody diluted 1:500 in blocking solution, followed by a goat anti-mouse antibody conjugated to Alexa488. Figure 2A: cells were immunofluorescently labelled with the 5-mC antibody (left) or with DAPI (right). Figure 2B and C: staining of the cells with the 5-mC antibody after incubation of the antibody with 50 μM mCTP or hmCTP, respectively.

    Fish

    Figure 3. FISH using the Diagenode monoclonal antibody directed against 5-mCTo detect methylated chromosomal regions, FISH was performed on metaphase chromosomes from HeLa cells using the Diagenode monoclonal antibody against 5-mC (Cat. No. C15200003). The cells were blocked in metaphase by treatment with colcemid (0.1 μg/ml) for 1 - 2 hours, fixed overnight at -20°C with ethanol/glacial acetic acid and treated with 2N HCl for 30’ at room temperature. Subsequently, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 and stained with the 5-mC antibody (left) diluted 1:1,000 in blocking solution, followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the chromosomes with DAPI. A merge of the two stainings is shown on the right.

    Fish

    Figure 4. FISH using the Diagenode monoclonal antibody directed against 5-mC
    To detect methylated chromosomal regions, FISH was performed on metaphase chromosomes from HeLa cells using the Diagenode monoclonal antibody against 5-mC (Cat. No. C15200003). The cells were blocked in metaphase by treatment with colcemid (0.1 μg/ml) for 1 - 2 hours, fixed overnight at -20°C with ethanol/glacial acetic acid and treated with 2N HCl for 30’ at room temperature. Subsequently, the cells were blocked with PBS containing 1% BSA and 0.1% Triton X-100 and stained with the 5-mC antibody (left) diluted 1:1,000 in blocking solution, followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the chromosomes with DAPI. A merge of the two stainings is shown on the right.

  • Applications
    Epigenetics DNA Methylation
    Complete solutions for DNA methylation studies Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data betwee... Read more
    DB
    Dot blotting Read more
    IF
    Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more
    FISH
    FISH Read more
  • Documents
    Datasheet 5-mC C15200003 DATASHEET
    Monoclonal antibody raised in mouse against 5-mC (5-methylcytosine) conjugated to BSA.
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    Antibodies you can trust POSTER
    Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...
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    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
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  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: 5-methylcytosine (5-mC) monoclonal antibody for ICC/IF - Classic (Diagenode Cat# C15200003 Lot# 001). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Transcriptome-wide distribution and function of RNA hydroxymethylcytosine
    Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F.
    Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, nota...

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