Anti-Dnmt3b 抗体 (ab2851)

Reviews (5)Q&A (12)References (76)

Key features and details

  • Rabbit polyclonal to Dnmt3b
  • Suitable for: IHC-P, WB
  • Reacts with: Mouse, Human
  • Isotype: IgG

製品の概要

  • 製品名

    Anti-Dnmt3b antibody
    Dnmt3b 一次抗体 製品一覧

  • 製品の詳細

    Rabbit polyclonal to Dnmt3b

  • 由来種

    Rabbit

  • 特異性

    This antibody detects DNA methyltransferase 3b (Dnmt3b) from human and mouse tissues and cells as well as recombinant human Dnmt3b. This antibody detects, to a lesser extent, full-length human recombinant Dnmt3a.

  • アプリケーション

    適用あり: IHC-P, WBmore details

  • 種交差性

    交差種: Mouse, Human

  • 免疫原

    Synthetic peptide corresponding to Mouse Dnmt3b aa 1-14.
    Sequence:

    MKGDSRHLNEEEGA


    (Peptide available as ab4922)

    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI

製品の特性

  • 製品の状態

    Liquid

  • 保存方法

    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.

  • バッファー

    Preservative: 0.05% Sodium azide
    Constituents: 0.1% BSA, 99% PBS
  • Concentration information loading...

  • 精製度

    Immunogen affinity purified

  • ポリ/モノ

    ポリクローナル

  • アイソタイプ

    IgG

  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab2851 in the following tested applications.

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

アプリケーション Abreviews 特記事項
IHC-P 1/100 - 1/1000.
WB Use a concentration of 2 µg/ml. Predicted molecular weight: 97.5 kDa.

ターゲット情報

  • 機能

    Required for genome wide de novo methylation and is essential for the establishment of DNA methylation patterns during development. DNA methylation is coordinated with methylation of histones. May preferentially methylates nucleosomal DNA within the nucleosome core region. May function as transcriptional co-repressor by associating with CBX4 and independently of DNA methylation. Seems to be involved in gene silencing (By similarity). In association with DNMT1 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. Isoforms 4 and 5 are probably not functional due to the deletion of two conserved methyltransferase motifs.

  • 組織特異性

    Ubiquitous; highly expressed in fetal liver, heart, kidney, placenta, and at lower levels in spleen, colon, brain, liver, small intestine, lung, peripheral blood mononuclear cells, and skeletal muscle. Isoform 1 is expressed in all tissues except brain, skeletal muscle and PBMC, 3 is ubiquitous, 4 is expressed in all tissues except brain, skeletal muscle, lung and prostate and 5 is detectable only in testis and at very low level in brain and prostate.

  • 関連疾患

    Defects in DNMT3B are a cause of immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) [MIM:242860]. ICF is a rare autosomal recessive disorder characterized by a variable immunodeficiency, mild facial anomalies, and centromeric heterochromatin instability involving chromosomes 1, 9, and 16. ICF is biochemically characterized by hypomethylation of CpG sites in some regions of heterochromatin.

  • 配列類似性

    Belongs to the C5-methyltransferase family.
    Contains 1 ADD domain.
    Contains 1 GATA-type zinc finger.
    Contains 1 PHD-type zinc finger.
    Contains 1 PWWP domain.

  • ドメイン

    The PWWP domain is essential for targeting to pericentric heterochromatin.

  • 翻訳後修飾

    Sumoylated.

  • 細胞内局在

    Nucleus.
  • Information by UniProt

  • 参照データベース

    • 別名

      • Cytosine 5methyltransferase 3B antibody
      • DNA antibody
      • DNA (cytosine 5) methyltransferase 3 beta antibody
      • DNA (cytosine 5)-methyltransferase 3B antibody
      • DNA (cytosine-5)-methyltransferase 3B antibody
      • DNA methyltransferase HsaIIIB antibody
      • DNA MTase HsaIIIB antibody
      • DNM3B_HUMAN antibody
      • Dnmt3b antibody
      • EC 2.1.1.37 antibody
      • ICF antibody
      • ICF1 antibody
      • M.HsaIIIB antibody
      • MGC124407 antibody
      • RP23-89H14.3 antibody
      see all

    画像

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3b antibody (ab2851)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3b antibody (ab2851)

      ab2851 labellling Dnmt3b in the nucleus of Mouse brain tissue (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS. Tissue sections were incubated with primary antibody (1:200 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

    • Western blot - Anti-Dnmt3b antibody (ab2851)
      Western blot - Anti-Dnmt3b antibody (ab2851)
      All lanes : Anti-Dnmt3b antibody (ab2851) at 1/1000 dilution

      Lane 1 : HeLa cell lysate
      Lane 2 : A549 cell lysate
      Lane 3 : NIH-3T3 cell lysate

      Lysates/proteins at 25 µg per lane.

      Predicted band size: 97.5 kDa
      Observed band size: 97 kDa
      why is the actual band size different from the predicted?

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3b antibody (ab2851)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3b antibody (ab2851)

      ab2851 labellling Dnmt3b in the nucleus of Human testis tissue (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS. Tissue sections were incubated with primary antibody (1:500 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3b antibody (ab2851)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dnmt3b antibody (ab2851)

      ab2851 labellling Dnmt3b in the nucleus and cytoplasm of Human breast carcinoma (right) compared with a negative control (left) by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, antigen retrieval method was performed using 10mM sodium citrate (pH 6.0) microwaved for 8-15 min. Tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS. Tissue sections were incubated with primary antibody (1:500 in 3% BSA-PBS) overnight at 4°C. A HRP-conjugated anti-rabbit was used as the secondary antibody, followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

    • Western blot - Anti-Dnmt3b antibody (ab2851)
      Western blot - Anti-Dnmt3b antibody (ab2851)
      All lanes : Anti-Dnmt3b antibody (ab2851) at 1 µg/ml

      Lane 1 : A498 (Human Kidney Carcinoma) Whole Cell Lysate
      Lane 2 : JEG-3 (Human placental choriocarcinoma cell line) Whole Cell Lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

      Developed using the ECL technique.

      Performed under reducing conditions.

      Predicted band size: 97.5 kDa
      Observed band size: 97.5 kDa
      Additional bands at: 32 kDa, 46 kDa. We are unsure as to the identity of these extra bands.


      Exposure time: 2 minutes

    参考文献 (76)

    ab2851 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

    ab2851 は 76 報の論文で使用されています。

    • Peng X  et al. Long non-coding RNA TNRC6C-AS1 promotes methylation of STK4 to inhibit thyroid carcinoma cell apoptosis and autophagy via Hippo signalling pathway. J Cell Mol Med 24:304-316 (2020). PubMed: 31657132
    • Shiah SG  et al. MiR-30a and miR-379 modulate retinoic acid pathway by targeting DNA methyltransferase 3B in oral cancer. J Biomed Sci 27:46 (2020). PubMed: 32238162
    • Guo X  et al. Long non-coding RNA-HAGLR suppressed tumor growth of lung adenocarcinoma through epigenetically silencing E2F1. Exp Cell Res 382:111461 (2019). PubMed: 31194977
    • Jin J  et al. Methylation-associated silencing of miR-128 promotes the development of esophageal cancer by targeting COX-2 in areas with a high incidence of esophageal cancer. Int J Oncol 54:644-654 (2019). PubMed: 30535495
    • Wu R  et al. Downregulation of KLF13 through DNMT1-mediated hypermethylation promotes glioma cell proliferation and invasion. Onco Targets Ther 12:1509-1520 (2019). PubMed: 30863117
    View all Publications for this product

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    1-10 of 17 Abreviews or Q&A

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-Dnmt3b antibody - ChIP Grade

     Good
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Muscle)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: EDTA 20 min at 97ºC in a PT link
    Permeabilization
    No
    Specification
    Muscle
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 25°C
    Fixative
    Formaldehyde
    Read More

    Abcam user community

    Verified customer

    投稿 Sep 24 2015

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Dnmt3b antibody - ChIP Grade

     Good
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (Cardiomyocytes)
    Specification
    Cardiomyocytes
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    投稿 Sep 18 2015

    Immunohistochemistry (PFA perfusion fixed frozen sections) abreview for Anti-Dnmt3b antibody - ChIP Grade

     Good
    Abreviews
    abreview image
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Sample
    Mouse Tissue sections (Brain)
    Permeabilization
    Yes - Triton x-100, 0.01%
    Specification
    Brain
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
    Fixative
    Paraformaldehyde
    Read More

    Abcam user community

    Verified customer

    投稿 Jun 29 2015

    Western blot abreview for Anti-Dnmt3b antibody - ChIP Grade

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (Cardiomyocytes)
    Gel Running Conditions
    Reduced Denaturing (10% PAGE)
    Loading amount
    25 µg
    Specification
    Cardiomyocytes
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    投稿 Jun 29 2015

    Question

    I'm using ab13604 in WB but seeing bands at 130 and 180 kDa and nothing at the expected 95 kDa. I would like to try ab2851.

    Read More
    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 ab2851. This will actually ship today and you should receive it tomorrow.

    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.

    Read More

    Question

    LOT NUMBER XXXX ORDER NUMBER XXXX DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE RAT NORMAL EMBRYO , total protein PRIMARY ANTIBODY Concentration or dilution 1:100-1:400 Diluent buffer 1:10 of blocking Incubation time O/N Incubation temperature 4°C Washing: Buffer Used PBST Number of washes 6 washes WITH 5 MINS CHANGE DETECTION METHOD ECL advance POSITIVE AND NEGATIVE CONTROLS USED Positive control NA Negative control NA ANTIBODY STORAGE CONDITIONS -20degC SAMPLE PREPARATION Lysis buffer RIPA Protease inhibitors COMPLETE PROTEASE Phosphatase inhibitors - Reducing agent DTT, MERCAPTO Boiling for ≥5 min? YES AMOUNT OF PROTEIN LOADED Protein loaded ug/lane or cells/lane 40ug ELECTROPHORESIS/GEL CONDITIONS Reducing or Non Reducing gel REDUCING Percentage of gel 10% Volts applied 100V Time applied 60-70 mins TRANSFER AND BLOCKING CONDITIONS Type of membrane NC Protein transfer verified YES Blocking agent and concentration 5% blocking provided in ECL advane kit( from amersham) Blocking time 1 hr Blocking temperature RT SECONDARY ANTIBODY Species Swine Reacts against Rabbit Concentration or dilution 1:10,000 Diluent buffer 1:10 of blocking Incubation time 1 hr Incubation temperature: RT Fluorochrome or enzyme conjugate HRP Washing: Buffer Used PBST Number of washes 10 washes WITH 5 MINS CHANGE HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Changing protein Concentration, blocking %, primary antibody dilution from 1:100 to 1: 400, secondary antibody dilution, washing time etc, ADDITIONAL NOTES The another antibody received with above mentioned (i.e. anti-SP3) worked fine with the same sample and same protocol indicating that there is no problem with the sample or protocol and secondary Ab.

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    Answer

    Thank you for contacting us and taking the time to send the questionnaire. We really appreciate all the details provided as well as the images sent, that help us to better understand the problem. All of our products are covered by our Abpromise guarantee; which ensures that you can trust our products, and they should work in the tested species and applications stated on the datasheet, or we will offer a replacement, credit, or refund, if reported within 6 months of purchase. I would really like to help you solve the problem and obtain good results from this antibody, and for that I am very pleased to give you some protocol tips. In the case these suggestions don’t improve the results, please let me know, and I will be happy to send a replacement or a credit note. -In order to enrich the signal, prepare nuclear lysates to increase the protein levels in the sample. -During sample preparation, it is crucial to use protease inhibitors as well as phosphatase inhibitors to avoid sample degradation. -I would suggest trying a different blocking agent. Have you tried 5%BSA for 1-2 hours at RT? -Decreasing the secondary concentration may also help to improve the signal. -To avoid smearing of the gel, using fresh prepared buffers is crucial. - You may run a positive control to assess how well the antibody is working. This protein is highly expressed in JEG-3 lysates. I hope these tips help, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again, and I’ll be more than happy to help you further.

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    Immunocytochemistry/ Immunofluorescence abreview for Anti-Dnmt3b antibody - ChIP Grade

     Below Average
    Abreviews
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Rat Cell (Hepatoma cells)
    Specification
    Hepatoma cells
    Fixative
    Formaldehyde
    Permeabilization
    Yes - 0.2% triton X-100
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    投稿 Jul 23 2008

    Question

    My apologies. I was not being clear. I need a good positive control target for anti-DNMT1 ChIP.

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    Answer

    A brief literature search revealed that PMID: 16357870 (Vire E et al Nature 2006 439:871) has used U2OS cells and primers specific for the hsMYT1 promoter. The primers are described in PMID: 15231737 (Kirmizis A et al Genes and Dev 2004 18:1592). I hope this information will be useful,

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    Question

    Our customer confused about the molecular weight you mentioned. They found that all referneces they searched showed that the molecular weight of this protein is ~130 kDa. The attachment file is the reference which were published in Vol. 439 at Feb 16 last year. This data is very important for our customer to carry on their research. We would be appreciated if you could help them elucidate this case, thank you!

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    Answer

    Thank you for getting back in touch with me. To clarify. A predicted molecular weight of 97KDa for Dnmt3b is derived from SwissProt reference Q9UBC3. This is purely based on the protein sequence and a prediction. ab2851 detects a band of approximately 130 kDa which correlates with the Dnmt3b reference that you have provided me with. I can tell you that this antibody also detects recombinant DNNMT3b1 at ~130KDa and recombinant DNMT3b3 at ~100 kDa; this may explain the additional bands. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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    Question

    I apologize for a delay in my response, but I didn't have a chance to reply before. I am sending the photo of the DNMT3B blot, I really don't know what happened with the prevous attachment. So, these are the bands that I am seeing when I use your antibody, upper one (which I believe is specific) is around let say 90 Kd and the lower (probably unspecific one) is around 80 Kda. And to answer your questions... Incubation with the primary antibody was overnight. And, no, I didn't try blocking with BSA with this Ab. I usually try that when I have very weak signal. In this case I have had pretty strong signal with very strong nonspecific band. So, I am sure background would be much more pronounced with BSA than with nonfat dry milk.I didn't find it reasonable to try when I already had very strong nonspecific band. Please let me know what you think about it.

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    Answer

    I have been advised by the source of ab2851 that it would be most important to run a positive control. In the lab recombinant DNMT3b has been used, however it is reported that DNMT3b is highly expressed in P19 (murine EC) cells when compared with human cells such as HeLa or HCT116. I would therefore recommend to run P19 cell lysate. It would be worth trying to block the membrane to see if the BSA alters the banding pattern. It would also be worth running a negative control (no primary antibody) to determine if the non-specific banding is being caused by interaction with the secondary antibody. An interesting point was raised by my colleagues: splice variants: It is known that there are at least 5 different splice variants for DNMT3b, and it is also believed that the protein is post-translationally modified. Therefore it is not unexpected to see some size heterogeneity. Finally, I may be able to add to the catalogue some neutralizing peptide to determine which bands are specific if you are interested in trying this. I hope the advice detailed above is useful, please do not hesitate to contact me if you have further questions,

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    1-10 of 17 Abreviews or Q&A

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