Overview

  • Product name
    Anti-5-methylcytosine (5-mC) antibody [5MC-CD]
    See all 5-methylcytosine (5-mC) primary antibodies
  • Description
    Mouse monoclonal [5MC-CD] to 5-methylcytosine (5-mC)
  • Host species
    Mouse
  • Specificity
    ab73938 reacts with DNA with 5-Methylcytosine (methylated DNA).
  • Tested applications
    Suitable for: Southern Blot, ICC/IF, Flow Cytmore details
  • Immunogen

    Chemical/ Small Molecule corresponding to 5-methylcytosine (5-mC) conjugated to Bovine Serum Albumin (BSA).

  • Positive control
    • Chlamidomonas me-1 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab73938 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
Southern Blot 1/1000.

Immunoblotting - detection of DNA with 5-methylocytosine on nitrocellulose. Use at 1/1000

ICC/IF 1/50 - 1/100. Recommend fixing in 4% PFA followed by denaturation with 70% formamide in 2x SSC and chilling in 70%, 95%, and 99% EtOH for 2 min each. Diluting antibody in SSC (saline sodium citrate) may give optimal results. See protocols from PubMed: 11983892 and 17994007
Flow Cyt 1/100.

ab91545 - Mouse monoclonal IgM, is suitable for use as an isotype control with this antibody.

 

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

  • ab73938 staining 5-Methyl Cytosine in human sperm cells by Flow Cytometry. Cells were fixed with 70% ethanol and permeabilized with 5% tween. The sample was incubated with the primary antibody (1/100 in PBST) for 30 minutes at 27°C. A Alexa Fluor® 488-conjugated goat anti-mouse IgG (1/300) was used as the secondary antibody.

     

    See Abreview

  • Methylation of chloroplast DNA visualized by immunochemistry. Samples are Chlamidomonas me-1 cells. Left: DAPI-stained cells. Middle: Cells stained with ab73938 and FITC-conjugated 2nd antibody. Right: Merged image. Chloroplast DNA is exclusively methylated in gamete cells
  • ab73938 staining 5-Methyl Cytosine in murine PN5 zygotes by Immunocytochemistry/ Immunofluorescence.

    Cells were fixed with methanol/acetic acid (3:1) and blocked with 30% serum for overnight a 4°C. Samples were incubated with primary antibody (1/100 in diluent) for 18 hours at 4°C before detection with a fluorescent secondary antibody.

References

This product has been referenced in:
  • Marycz K  et al. Evaluation of Oxidative Stress and Mitophagy during Adipogenic Differentiation of Adipose-Derived Stem Cells Isolated from Equine Metabolic Syndrome (EMS) Horses. Stem Cells Int 2018:5340756 (2018). Read more (PubMed: 29977307) »
  • Kornicka K  et al. The effects of the DNA methyltranfserases inhibitor 5-Azacitidine on ageing, oxidative stress and DNA methylation of adipose derived stem cells. J Cell Mol Med 21:387-401 (2017). Read more (PubMed: 27998022) »
See all 18 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Application
Flow Cytometry
Fixation
ethanol 70%
Permeabilization
Yes - tween 5%
Sample
Human Cell (sperm)
Specification
sperm
Gating Strategy
10000
Preparation
Cell harvesting/tissue preparation method: percolgradients
Sample buffer: PBS

Ms. Jaleh Barzideh

Verified customer

Submitted Dec 23 2013

Answer

Thank you for contacting us.

For the use of ab73938in ICC/IF,I would recommendthe following secondary antibodies:
DyLight 488: ab97007,https://www.abcam.com/ab97007
DyLight 549: ab97008, https://www.abcam.com/ab97008
DyLight 594: ab97009, https://www.abcam.com/ab97009
DyLight 649: ab97010, https://www.abcam.com/ab97010

Theses are some links you may find useful to design your ICC/IF experiments:
https://www.abcam.com/index.html?pageconfig=resource&rid=11417
https://docs.abcam.com/pdf/general/understanding_secondary_antibodies.pdf
https://www.abcam.com/index.html?pageconfig=resource&rid=12401

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton-X100 in PBS

Dr. Kirk Mcmanus

Verified customer

Submitted Feb 07 2012

Answer

Thank you for your inquiry. We already removed Southern Blot as application as well as Western Blot. Both applications are somewhat inappropriate since the antibody target is not protein but DNA with methylated cytosine. But I am happy to confirm that this antibody can be used for "immunoblotting" as published by Sano et al. Please review the specific references section of the datasheet for more publications. If you have used this antibody before, we would like to encourage you to submit an Abreview. You would earn Abpoints that can be redeemed as discount on future purchases or Amazon gift vouchers. If you are planning to purchase this antibody shortly, please let me know since you might be eligible to take part in our "supply an image" discount program. I hope this information is helpful. please do not hesitate to contact me again with any further questions or concerns.

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Answer

Thanks you for sending the information on you IHC protocol. I am still trying to see if I can find any IHC protocols that are better than others for detected DNA modifications. If I find any I will send them on as soon as I find them. In the meantime, I have attached our general IHC guide, which contains information on several antigen retrieval methods. If there is anything else I can help you with, please let me know.

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Question
Answer

Thank you for your call today and for letting us know about the trouble with ab73938. As we discussed, please keep me updated of any new results using the antibody at 1:50, and we would appreciate it very much if you send an image of the staining. I look forward to hearing from you. Please let me know if you have any questions.

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Answer

Thank you for your enquiry. The specificity of the anti-5Methylcytosine antibody has been tested in many published papers included articles by Prof. H. Sano who has done a lot of work on this antibody. Some of them were cited in the references in the datasheet. I recommend to review these for further information. Further references would be available by PubMed search with key words 'Sano H, 5Methyytosine', or 'DNA methylation'. From recent studies, methylated cytosine protrude from the duplex, therefore the antibody may be able to recognize metylation on both native duplex and denatured DNA. I am sorry we have no additional data at the moment. From the online datasheet: Sano H et al. Detection of heavy methylation in human repetitive DNA subsets by a monoclonal antibody against 5-methylcytosine. Biochim Biophys Acta 951:157-65 (1988). PubMed: 2847796 Additionally: Sano H et al “Identification of 5-methycytosine in DNA fragment immobilized on nitrocellulose paper “PNAS 77:3581-3585 (1980) PMID: 6251470 I hope this will be helpful to you. Should you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for contacting us and sorry for the delay in getting back to you.


The immunogen used to raise Anti-5-Methyl Cytosine antibody [5MC-CD] (ab73938) is 5-methyl Cytosine conjugated to BSA. It is not 5-methyl Cytosine as part of a CpG dinucleotide.


I hope this information has been of help. If you require any further information please do not heitate to contact us again.

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

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