Overview

  • Product name
    Anti-Dnmt3b antibody - ChIP Grade
    See all Dnmt3b primary antibodies
  • Description
    Rabbit polyclonal to Dnmt3b - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    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.
  • Tested applications
    Suitable for: ICC/IF, IP, IHC-P, WB, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

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

    MKGDSRHLNEEEGA


    (Peptide available as ab4922)

  • Positive control
    • P19 nuclear extracts. ChIP: 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).

Properties

Applications

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.

Application Abreviews Notes
ICC/IF Use a concentration of 1 µg/ml.
IP Use at an assay dependent concentration. PubMed: 16631596
IHC-P 1/100 - 1/1000.
WB Use a concentration of 2 µg/ml. Detects a band of approximately 130 kDa (predicted molecular weight: 97.5 kDa).Can be blocked with Dnmt3b peptide (ab4922).
ChIP Use at an assay dependent concentration. PubMed: 17972916

Target

  • Function
    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.
  • Tissue specificity
    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.
  • Involvement in disease
    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.
  • Sequence similarities
    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.
  • Domain
    The PWWP domain is essential for targeting to pericentric heterochromatin.
  • Post-translational
    modifications
    Sumoylated.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • 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

Images

  • 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.

  • All lanes : Anti-Dnmt3b antibody - ChIP Grade (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?

  • 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.

  • ICC/IF image of ab2851 stained HepG2 cells. The cells were 100% methanol fixed (5 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 (ab2851, 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.

  • 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.

  • All lanes : Anti-Dnmt3b antibody - ChIP Grade (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

References

This product has been referenced in:
  • Altshuler A  et al. RAS Regulates the Transition from Naive to Primed Pluripotent Stem Cells. Stem Cell Reports 10:1088-1101 (2018). WB ; Mouse . Read more (PubMed: 29456180) »
  • Pan XY  et al. DNA Methylation of PTGIS Enhances Hepatic Stellate Cells Activation and Liver Fibrogenesis. Front Pharmacol 9:553 (2018). Read more (PubMed: 29892223) »
See all 46 Publications for this product

Customer reviews and Q&As

1-10 of 17 Abreviews or Q&A

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

Abcam user community

Verified customer

Submitted Sep 24 2015

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

Abcam user community

Verified customer

Submitted Sep 18 2015

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

Abcam user community

Verified customer

Submitted Jun 29 2015

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

Abcam user community

Verified customer

Submitted Jun 29 2015

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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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

Abcam user community

Verified customer

Submitted Jul 23 2008

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,

Read More

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.

Read More

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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