Anti-5-methylcytosine (5-mC) antibody [33D3] (ab10805)

Mouse monoclonal 5-methylcytosine (5-mC) antibody [33D3]. Validated in IP, IHC, SB, Flow Cyt, ChIP, Dot, MeDIP. Cited in 67 publication(s). Independently reviewed in 12 review(s).

Overview

  • Product name
    Anti-5-methylcytosine (5-mC) antibody [33D3]
    See all 5-methylcytosine (5-mC) primary antibodies
  • Description
    Mouse monoclonal [33D3] to 5-methylcytosine (5-mC)
  • Host species
    Mouse
  • Specificity
    Clone 33D3 has been developed to discriminate between the modified base 5-MeCyd and the normal counterpart cytosine.
  • Tested applications
    Suitable for: IHC-P, MeDIP, ChIP, IP, Southern Blot, Dot blot, Flow Cyt, IHC-Frmore details
    Unsuitable for: ICC
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Chemical/ Small Molecule corresponding to 5-methylcytosine (5-mC).

  • General notes

    Storage in frost-free freezers is not recommended. Should this product contain a precipitate microcentrifugation before use.While older lots have performed well in ICC, we have received inconsistent results with the latest lots. Unfortunately, we can no longer guarantee this antibody for use in ICC.

     

     

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.4
    Preservative: 0.01% Thimerosal (merthiolate)
    Constituent: 99.99% PBS
  • Concentration information loading...
  • Purity
    Protein A purified
  • Primary antibody notes
    Ab10805 recognises the modified base 5-methylcytidine found in DNA of plants and vertebrates. DNA methylation is a DNA modification process, which is involved in the control of gene expression. Reports suggest that in tumours, DNA is frequently globally hypomethylated compared to the DNA from normal tissue.
  • Clonality
    Monoclonal
  • Clone number
    33D3
  • Myeloma
    Sp2/0-Ag14
  • Isotype
    IgG1
  • Light chain type
    kappa
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab10805 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
MeDIP Use at an assay dependent concentration. PubMed: 17845077Used 5ul in 200ul reaction with 2ug digested genomic DNA from Drosophila.
ChIP Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
Southern Blot 1/200.
Dot blot Use at an assay dependent concentration. PubMed: 24386123
Flow Cyt Use at an assay dependent concentration.

Use 10ul of working dilution to label 1000000 cells in 100ul. (see Habib, M. et al. (1999)).
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-Fr Use at an assay dependent concentration.
  • Application notes
    Is unsuitable for ICC.
  • Target

    • Form
      The native context of double-stranded DNA may obstruct antibody binding to 5-methylcytosine. For successful detection of 5-methylcytosine, we recommend that the DNA is denatured to make the nucleotides accessible for the antibody. Denaturing methods vary depending on each application.
    • Alternative names
      • 5 m C antibody
      • 5 mC antibody
      • 5 me C antibody
      • 5 Me Cytidine antibody
      • 5 Me Cytosine antibody
      • 5 meC antibody
      • 5 Methycytosine antibody
      • 5 Methyl Cytidine antibody
      • 5 Methyl Cytosine antibody
      • 5 Methyl-Cytidine antibody
      • 5 Methyl-Cytosine antibody
      • 5 MethylCytidine antibody
      • 5 MethylCytosine antibody
      • 5-mC antibody
      • 5-Me Cytidine antibody
      • 5-Me Cytosine antibody
      • 5-meC antibody
      • 5-Methyl Cytidine antibody
      • 5-Methyl-Cytidine antibody
      • 5-Methyl-Cytosine antibody
      • 5-MethylCytidine antibody
      • 5-MethylCytosine antibody
      • 5mC antibody
      • methyl CpG antibody
      see all

    Images

    • ab10805 staining 5-Methyl Cytidine in pig embryo (15 to 17 days) tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent fixation in paraformaldehyde, heat mediated antigen retrieval in Tris-EDTA buffer, permeabilization in Triton X-100 and blocking in 2% BSA for 10 minutes at 25°C. The primary antibody was diluted, 1/100 (PBS + 2% BSA) and incubated with sample for 1 hour at 25°C. An Alexa Fluor® 488 conjugated donkey polyclonal to mouse at 1/250 dilution, was used as secondary.

      See Abreview

    • Native ChIP analysis using unpurified ab10805 binding 5-methylcytosine (5-mC) in nuclear cell lysate derived from mouse embryonic stem cells. Samples were incubated with primary antibody (2µg/µg chromatin) for 16 hours at 4°C in a Glycerol IP buffer. Protein binding was detected using real-time PCR.

      Positive control: Magoh2 in HMT KO cells.

      Negative Control: Magoh2 in WT cells.

      See Abreview

    • Dot blot carried out using ab10805 (left blot), showing specificity to 5-methylcytosine. Indicated amounts (20–5 ng) of oligonucleotides containing either 5mC (50%) or 5hmC (20 %) were spotted on positive charged nylon membrane and detected either with 5mC (ab10805) or 5hmC antibody. 

    References

    This product has been referenced in:
    • Abukiwan A  et al. Dexamethasone-induced inhibition of miR-132 via methylation promotes TGF-ß-driven progression of pancreatic cancer. Int J Oncol 54:53-64 (2019). Read more (PubMed: 30387838) »
    • Selli C  et al. Molecular changes during extended neoadjuvant letrozole treatment of breast cancer: distinguishing acquired resistance from dormant tumours. Breast Cancer Res 21:2 (2019). Read more (PubMed: 30616553) »
    See all 70 Publications for this product

    Customer reviews and Q&As

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    1-10 of 12 Abreviews

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Lung bronchial biopsy)
    Antigen retrieval step
    None
    Permeabilization
    No
    Specification
    Lung bronchial biopsy
    Blocking step
    20% FCS, 1% BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 25°C
    Fixative
    GMA-embedded

    Abcam user community

    Verified customer

    Submitted May 23 2018

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (18dpc Male Urogenital Sinus)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citrate, pH 6
    Permeabilization
    No
    Specification
    18dpc Male Urogenital Sinus
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Dec 28 2017

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Flow Cytometry
    Sample
    Zebrafish Cell (Dissociated tissue cells)
    Permeabilization
    Yes - Methanol 90%
    Gating Strategy
    Cells were gated according to their DNA content
    Specification
    Dissociated tissue cells
    Preparation
    Cell harvesting/tissue preparation method: Tissue were dissociated with collagenase. Obtained cells were filtered and fixed.
    Sample buffer: Cells were analysed in PBS
    Fixation
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Aug 01 2017

    Application
    Other
    Sample
    Zebrafish Tissue lysate - other (5 dpf embryo)

    Kathryn Bambino

    Verified customer

    Submitted Feb 03 2015

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunoprecipitation
    Sample
    Rat Tissue lysate - other (mammary gland)
    Total protein in input
    20 µg
    Specification
    mammary gland
    Treatment
    maternal high fat
    Immuno-precipitation step
    Other - Dynabeads

    Abcam user community

    Verified customer

    Submitted Feb 13 2013

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    ChIP
    Sample
    Mouse Cell lysate - nuclear (Mouse ES cells)
    Negative control
    Magoh2 in WT cells
    Specification
    Mouse ES cells
    Detection step
    Real-time PCR
    Type
    Native ChIP (N-ChIP)
    Positive control
    Magoh2 in HMT KO cells

    Abcam user community

    Verified customer

    Submitted Dec 03 2012

    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Human glioma cells)
    Specification
    Human glioma cells
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.3% Triton X-100
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Nov 11 2011

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    ChIP
    Sample
    Human Cell lysate - nuclear (Human glioma cell DNA)
    Specification
    Human glioma cell DNA
    Type
    Native ChIP (N-ChIP)
    Detection step
    Semiquantitative PCR
    Positive control
    5hmC Ab
    Negative control
    IgG

    Abcam user community

    Verified customer

    Submitted Aug 01 2011

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (brain)
    Specification
    brain
    Fixative
    Formaldehyde
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: 10 mM Citrate buffer
    Permeabilization
    Yes - 0.3% Triton
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Jul 13 2011

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Pig Tissue sections (embryos of age 15 to 17 days)
    Specification
    embryos of age 15 to 17 days
    Fixative
    Paraformaldehyde
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Tris-EDTA
    Permeabilization
    Yes - triton X 100
    Blocking step
    BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 25°C

    Ms. Sara Maj Wätjen Hyldig

    Verified customer

    Submitted Mar 05 2009

    1-10 of 12 Abreviews

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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