Overview

  • Product name
    Anti-Dnmt1 antibody [60B1220.1] - ChIP Grade
    See all Dnmt1 primary antibodies
  • Description
    Mouse monoclonal [60B1220.1] to Dnmt1 - ChIP Grade
  • Host species
    Mouse
  • Specificity
    This antibody detects a ~180 kDa protein, corresponding to the apparent molecular mass of Dnmt1 on SDS-PAGE immunoblots in samples of human and mouse origin. Immunogen itself has been shown to be toxic.
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, ChIP, Flow Cyt, WB, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Zebrafish
    Predicted to work with: Cow
  • Immunogen

    Synthetic peptide:

    EKDDREDKENAFKR

    , corresponding to amino acids 637-650 of Human Dnmt1

  • Positive control
    • IHC-P: Mouse brain and kidney medullar tissue. Flow cyt: HeLa cells. WB: HCT 116 cells; Dnmt1 recombinant protein. Human kidney (IHC), mouse ES or NIH 3T3 cell lysate (WB)
  • General notes
    For maximum product recovery, centrifuge the product vial before removing cap. Shipped at on gel packs.

Properties

Applications

Our Abpromise guarantee covers the use of ab13537 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 a concentration of 1 - 2 µg/ml. DNMT1 high levels are toxic, as a result it may be tough to find sometimes. Signal amplification might be needed.
IHC-Fr Use at an assay dependent concentration.
ChIP Use at an assay dependent concentration.
Flow Cyt 1/100.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

WB Use a concentration of 2 - 4 µg/ml. Detects a band of approximately 180 kDa (predicted molecular weight: 183 kDa).
IP Use 2µg for 106 cells.

Target

  • Function
    Methylates CpG residues. Preferentially methylates hemimethylated DNA. Associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand, that is essential for epigenetic inheritance. Associates with chromatin during G2 and M phases to maintain DNA methylation independently of replication. It is responsible for maintaining methylation patterns established in development. DNA methylation is coordinated with methylation of histones. Mediates transcriptional repression by direct binding to HDAC2. In association with DNMT3B and via the recruitment of CTCFL/BORIS, involved in activation of BAG1 gene expression by modulating dimethylation of promoter histone H3 at H3K4 and H3K9.
  • Tissue specificity
    Ubiquitous; highly expressed in fetal tissues, heart, kidney, placenta, peripheral blood mononuclear cells, and expressed at lower levels in spleen, lung, brain, small intestine, colon, liver, and skeletal muscle. Isoform 2 is less expressed than isoform 1.
  • Sequence similarities
    Belongs to the C5-methyltransferase family.
    Contains 2 BAH domains.
    Contains 1 CXXC-type zinc finger.
  • Domain
    The N-terminal part is required for homodimerization and acts as a regulatory domain.
  • Post-translational
    modifications
    Sumoylated; sumoylation increases activity.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • ADCADN antibody
    • AIM antibody
    • CXXC finger protein 9 antibody
    • CXXC-type zinc finger protein 9 antibody
    • CXXC9 antibody
    • DNA (cytosine 5 ) methyltransferase 1 antibody
    • DNA (cytosine-5)-methyltransferase 1 antibody
    • DNA methyltransferase 1 antibody
    • DNA methyltransferase HsaI antibody
    • DNA methyltransferase M.HsaI. antibody
    • DNA MTase antibody
    • DNA MTase HsaI antibody
    • DNMT 1 antibody
    • DNMT antibody
    • Dnmt1 antibody
    • DNMT1_HUMAN antibody
    • Dnmt1o antibody
    • FLJ16293 antibody
    • HSN1E antibody
    • M.HsaI antibody
    • MCMT antibody
    • Met1 antibody
    • MGC104992 antibody
    • mMmul antibody
    • MommeD2 antibody
    see all

Images

  • Anti-Dnmt1 antibody [60B1220.1] - ChIP Grade (ab13537) at 1/1000 dilution + 0.1ug mouse recombinant protein.
    Additional lower band is probably degraded or in other ways modified Dnmt1.

    Predicted band size: 183 kDa

  • ab13537 at 1/10000 dilution staining Dnmt1 in mouse brain tissue section by Immunohistochemistry (Bouin's fixative fixed paraffin-embedded tissue sections). Tissue underwent heat mediated antigen retrieval in microwave with two, 5 minutes incubation intervals in citrate buffer. An antibody amplifier™ was used for staining. A HRP-conjugated anti-mouse secondary was used at 1/10 dilution.

  • Overlay histogram showing HeLa cells stained with ab13537 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab13537, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • Mouse medullar kidney tissue stained for Dnmt1 using ab13537 at 1/1000 dilution in immunohistochemical analysis. DAB staining. Counterstained with hematoxylin.

  • All lanes : Anti-Dnmt1 antibody [60B1220.1] - ChIP Grade (ab13537) at 1 µg/ml

    Lane 1 : HCT116 whole cell lysate
    Lane 2 : HCT116 DNMT1 KO whole cell lysate
    Lane 3 : HCT116 DNMT3b KO whole cell lysate

    Secondary
    All lanes : HRP conjugated Sheep anti-mouse IgG

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 183 kDa


    Exposure time: 1 minute

    See Abreview

References

This product has been referenced in:
  • Zhang X  et al. The inhibition of UBC13 expression and blockage of the DNMT1-CHFR-Aurora A pathway contribute to paclitaxel resistance in ovarian cancer. Cell Death Dis 9:93 (2018). Read more (PubMed: 29367628) »
  • Zhao M  et al. IL-6/STAT3 pathway induced deficiency of RFX1 contributes to Th17-dependent autoimmune diseases via epigenetic regulation. Nat Commun 9:583 (2018). ChIP . Read more (PubMed: 29422534) »
See all 81 Publications for this product

Customer reviews and Q&As

1-10 of 20 Q&A

Answer

Thank you for contacting us.

I can recommend the following secondary antibodies, which are enzyme conjugated to HRP that would be suitable for the detection of the three primary antibodies (Anti-Dnmt3b antibody [52A1018] - ChIP Grade (ab13604); Anti-Dnmt1 antibody [60B1220.1] - ChIP Grade and Anti-Dnmt3a antibody [64B1446] - ChIP Grade (ab13888) :

https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Mouse-IgG1-heavy-chain-HRP-pre-adsorbed-ab98693.html

https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Mouse-IgG1-heavy-chain-HRP-ab97240.html

If you would prefer another conjugate please let me know and I will try to find the right one for you.
I hope this information is helpful. Please do not hesitate to contact me again if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question
Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1208729 (ab92453).

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

Thank you for your email.

We have decided to send you a replacement antibody. The antibody ab92453 is of same clone and same immunogen sequence. Could you check you and confirm?

Please note, although ab13537 and ab92453 seems same however they are developed for us by different laboratories so we can expect some variations as different preparations of the hybridoma are known to alter epitope specificity slightly.

Please also note that as of ab13537 this antibody has also not been tested in double staining where the second antibody was against same antigen. So protocol optimizations are highly recommended.

I will look forward to hearing from you soon. Please do not hesitate to contact me if you have any question.

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Question
Answer

Thank you for sending the details.

I am sorry without an image it is difficult to understand what is going on with staining. From description of the problem I can sense that the polyclonal antibody might be giving non-specific staining or high background - this is common with polyclonal antibodies as they need more optimization than monoclonal products. Also in double immunostaining one of the requirement is to use the individually optimized antibodies so I would strongly suggest to try these antibodies individually and observing the results. Once the satisfactory results are achieved, double immunostaining can then be performed using same conditions.

The immunohistochemistry on paraffin embedded sections is quite tricky,, in most cases antigen retrieval method needs further optimization.

It will also be good to verify the specificity of secondary antibodies to their respective primaries.

We also haven't tried these antibodies in double immunostaining so the conditions has to be determined by end user.

I hope these suggestions will be helpful. Please let me know if there is no improvements we will be happy to assist further.

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Answer

Thank you for contacting us.

If there is no other protocol then our standard protocols suggested on the product data sheet, the general protocols should be sufficient. However, I contacted the laboratory to get the protocol that was used doing the testing of the antibody. The protocol used is as followed:

Immunohistochemistry protocol for Antibody detection in formalin fixed tissues for Antibody Detection in Formalin-fixed for Antibody Tissues

Tissue Preparation: Formalin fixation and embedding in paraffin wax

Tissue Sectioning: Make 4-μm sections and place on pre-cleaned and charged microscope slides.

Heat in a tissue-drying oven for 45 minutes at 60°C.

Deparaffinization: Wash dry slides in 3 changes of xylene – 5 minutes each @ RT.

Rehydration: Wash slides in 3 changes of 100% alcohol – 3 minutes each @ RT.

Wash slides in 2 changes of 95% alcohol – 3 minutes each @ RT.

Wash slides in 1 change of 80% alcohol – 3 minutes @ RT.

Rinse slides in gentle running distilled water – 5 minutes @ RT.

Antigen retrieval: Steam slides in 0.01 M sodium citrate buffer, pH 6.0 at 99-100°C - 20 minutes.

Remove from heat and let stand at room temperature in buffer - 20 minutes.

Rinse in 1X TBS with Tween (TBST) – 1 minute @ RT.

Immunostaining: Do not allow tissues to dry at any time during the staining procedure

Apply a universal protein block – 20 minutes @ RT.

Drain protein block from slides, apply diluted primary antibody – 45 minutes @ RT.

Rinse slides in 1X TBST - 1 minute @ RT.

Apply a biotinylated anti-rabbit IgG (H+L) secondary – 30 minutes @ RT.

Rinse slides in 1X TBST - 1 minute @ RT.

Apply alkaline phosphatase streptavidin – 30 minutes @ RT.

Rinse slides in 1X TBST - 1 minute @ RT.

Apply alkaline phosphatase chromogen substrate – 30 minutes @ RT.

Wash slides in distilled water – 1 minute @ RT.

Dehydrate: This method should only be used if the chromogen substrate is alcohol

insoluble (e.g. Vector Red, DAB)

Wash slides in 2 changes of 80% alcohol – 1 minute each @ RT.

Wash slides in 2 changes of 95% alcohol – 1 minute each @ RT.

Wash slides in 3 changes of 100% alcohol – 1 minute each @ RT.

Wash slides in 3 changes of xylene – 1 minute each @ RT.


Apply coverslip.
Please be aware that any protocol may need optimisation for each experiment carried out.

Regarding the question for a negative control, according to "The Human Protein Atlas" Dnmt1 seems not to be expressed in for example in CNS (brain) and Liver/Pancreas. You might want to look at the webpage http://www.proteinatlas.org/ENSG00000130816 and confirm the information by some literature search.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for your email. I am sorry to hear that you have been experiencing problems with this antibody.

I have read the details you have kindly provided and have following further questions for better understanding of the problem;

- What type of cells are being used? Are they treated or non treated?
- Could you fill in the attached questionnaire?
- Were the conditions e.g. concentration and dilution optimized to be used in double IF?
- Could you provide image with all the details e.g. with secondary info, conjugate info etc?
- Could you high light the cells that concerns you or provide images in support?

Thank you very much for your cooperation. I will look forward to hearing from you soon.

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Answer

Thank you very much for contacting us with your enquiry.

The Dnmt1 antibody, ab13537, has been tested with human samples and in Western blot, and we fully guarantee the results of the antibody in the assay you will be performing.

Our AbTrial testing program is applicable when you'd like to test a product in a species or application that we have not tested previously. Since we have tested the antibody in your assay and guarantee the results, the testing program does not apply.

I hope that this will be helpful, but please let me know if you have any questions or if there is anything else that we can do for you.

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Answer

Thank you for contacting us.
We unfortunately haven't tested this antibody with Rat species so we are unable to confirm it. The immunogen show 78% homology with Rat sequence and because the antibody is monoclonal so we would not be able to comment on cross reactivity without checking the tested data. You can however try the antibody with rat samples along with your mouse samples.
Please click the following link for homology check;
http://www.ebi.ac.uk/Tools/services/web_clustalw2/toolresult.ebi?tool=clustalw2&jobId=clustalw2-I20120622-105429-0516-59353343-oy
I hope this information is nevertheless helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you very much for your interest in ab13537.
To our knowledge, ab13537 has not been tested in rat, however the immunogen of the antibody shares 92% sequence homology with rat and should cross-react. However, this is not tested and therefore not guaranteed. Therefore, I can offer a discount off a future purchase if you buy ab13537 now, test it in rat and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free primary antibody off your next Abcam order.
If you are interested in this offer, please follow these steps:
1. Reply to this e-mail to let me know that you would like to proceed and test ab13537 in rat. I will then send a discount code. This code must be issued before purchasing ab13537 so please wait for my reply before ordering.
2. Purchase ab13537 either by phone, fax, or online (www.abcam.com).
3. Test it in rat.
4. Let us know the results, positive or negative, using our Abreview system (this will take about 10 minutes and images are great if you have them!). To find out how to submit an Abreview, please visit: https://www.abcam.com/abreviews.
5. After the review is submitted to us, the discount code becomes active. Simply place your new order by phone, fax, or on the web and mention the discount code. The discount can be redeemed for any primary antibody ordered and the discount code is valid for 4 months after issue.
We are always pleased to obtain feedback about our products and any information is greatly appreciated! Even if ab13537 turns out to be unsuitable for rat, you will still receive the discount on your next purchase after your Abreview has been submitted.
Please let me know if you have any questions about this offer and I would be happy to help you further.
The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount.

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Answer

Thanks for re-sending your western blot images and for providing the extraction buffer information.
I see what you are saying. Interestingly, it seems there is background staining in some but not all of the murine hepatocyte samples. Are there any differences in the samples that might explain this?

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1-10 of 20 Q&A

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