Overview

  • Product name
  • Description
    Rabbit polyclonal to Dnmt1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IF, IP, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 100 - 200 of Human Dnmt1.

    Read Abcam's proprietary immunogen policy (Peptide available as ab21999.)

  • Positive control
    • This antibody gave a positive signal in: (WB) HeLa nuclear; (IF) Hek293; HepG2; MCF7
  • General notes
    The immunogen used to generate this antibody has 71% identity with the corresponding region in mouse Dnmt1. We have had variable feedback about the ability of this antibody to recognise mouse Dnmt1. Customers may prefer to use one of our other antibodies for detection of mouse Dnmt1

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
    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab19905 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
WB Use a concentration of 1 µg/ml. Detects a band of approximately 183 kDa (predicted molecular weight: 183 kDa).
ICC/IF Use a concentration of 1 µg/ml.
IP Use at an assay dependent concentration.
IHC-P Use a concentration of 1 - 10 µg/ml.

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

  • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: DNMT1 knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)
    Lane 4: Hek293 whole cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab19905 observed at 170 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab19905 was shown to recognize DNMT1 when DNMT1 knockout samples were used, along with additional cross-reactive bands. Wild-type and DNMT1 knockout samples were subjected to SDS-PAGE. Ab19905 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10010 dilution for 1 hour at room temperature before imaging.

  • All lanes : Anti-Dnmt1 antibody (ab19905) at 1 µg/ml

    Lane 1 : HeLa nuclear lysate
    Lane 2 : HeLa nuclear lysate with Human Dnmt1 peptide (ab21999) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 183 kDa
    Observed band size: 183 kDa



    ab19905 specifically recognises Dnmt1 at 183 kDa in HeLa nuclear extracts (lane1), which is efficiently blocked using the immunising peptide (ab21999) (lane2).
  • Dnmt1 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Dnmt1 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab19905.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: 183kDa: Dnmt1.
  • ab19905 at a 1/200 dilution staining paraformaldehyde fixed asynchronous HeLa cells by ICC/IF. The antibody was incubated with the cells for 30 minutes and then bound antibody was detected using a Cy3 conjugated goat anti-rabbit antibody. Nuclei were visualised using DAPI staining. Ab19905 gives a pattern that is predominantly enriched within nuclei.

    This image is courtesy of an Abreview submitted by Kirk McManus.

    See Abreview

  • Staining of human tonsil tissue was performed using ab19905. Strong nuclear staining was observed in germinal center cells and scattered cells in the interfollicular area. T cells were weakly to moderately positive and endothelial cells were positive.
  • ab19905 (1/100) staining Dnmt1 in paraffin-embbeded mouse testis tissue sections. Tissue underwent fixation in formaldehyde, heat-mediated antigen retrieval in 10mM citrate buffer pH 6.0 and blocking (30 minutes, 8% milk). A biotinylated anti-rabbit IgG (H+L) antibody was used as the secondary. For further experimental details please refer to abreview.

    See Abreview

  • Jurkat cells were incubated at 37°C for 5 days with vehicle control (0 µM) and different concentrations of hydralazine hydrochloride (ab120863). Decreased expression of DNMT1 (ab19905) in Jurkat cells correlates with an increase in hydralazine hydrochloride concentration, as described in literature.

    Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab19905 at 1 µg/ml and ab8227 at 1 µg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

  • ICC/IF image of ab19905 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab19905, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 1% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 1µg/ml.

References

This product has been referenced in:
  • Fudhaili A  et al. Resveratrol epigenetically regulates the expression of zinc finger protein 36 in non-small cell lung cancer cell lines. Oncol Rep 41:1377-1386 (2019). Read more (PubMed: 30535453) »
  • Chen J  et al. Curcumin-induced promoter hypermethylation of the mammalian target of rapamycin gene in multiple myeloma cells. Oncol Lett 17:1108-1114 (2019). Read more (PubMed: 30655872) »
See all 36 Publications for this product

Customer reviews and Q&As

1-10 of 10 Q&A

Question
Answer

Could you send us the new purchase order number so that we can process the order for ab199905?

Thanks!

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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 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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Question
Answer

Thank you very much for your call today and for letting us know about the trouble with ab19905 in Western blot.
Please keep me updated about the results after trying the 1 ug/mL concentration, and if you need any further assistance I'll be happy to help.
I look forward to hearing from you. Please let me know if you have further questions or need anything else at this time.

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Answer

Thank you for contacting us.

We will guarantee our antibodies for any species and applications listed as tested on our datasheets, even if the reviews are negative. If for any reason you are unsatisfied with your results when using one of our antibodies in a tested species or application, we are more than happy to refund or replace it for you.

If you are interested in testing any of our antibodies in an untested application, you may qualify for our Abtrial program:

https://www.abcam.com/Abtrial

If you purchase the antibody for full price and submit an Abreview with your protocol and results, we will provide a discount code that can be redeemed for your original purchase price off of a future order.

In general, ChIP tends to be the more sensitive application, and antibodies that work in ChIP will be more likely to work in WB than the other way around. I hope this helps, please let me know if you need any additional information or assistance.

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Answer

Thank you for contacting us.

The antibody ab19005 failed ChIP testing in March. I recommend ab13537 for human, mouse, and zebrafish samples.

Two other Dnmt1 antibodies that have been validated for ChIP are ab92453 and ab87656.

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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Answer

Gracias por la espera.

Este anticuerpo se testa de rutina en lisados celulares de IOUD2 (célula madre embriónica de ratón), pero no se ha usado para immunohistoquímica, por lo que toda la información referente al uso del anticuerpo en ratón mediante immunohistoquímica es la disponible en la web, procedente de la publicación de un usuario.

De todas formas, existe un porcentaje de homología del 71% entre las especies, que no es tan elevado como para afirmar que pueda reconocer la proteína en ratón, a partir del 85% es una buena estimación para confirmar la reactividad.

Espero que esta información te haya aclarado las dudas. Si no es así, por favor, no dudes en volvernos a contactar.

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Answer

Thank you for your enquiry. The antigen retrieval solution that was used for the testing of rabbit polyclonal to Dnmt1 (ab19905) was Tris-EDTA pH9. This was a standard preparation as follows; Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0). For further details please see http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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