概述

  • 产品名称
  • 描述
    兔多克隆抗体to Dnmt3b
  • 宿主
    Rabbit
  • 经测试应用
    适用于: WB, IHC-P, ICC/IFmore details
  • 种属反应性
    与反应: Human
  • 免疫原

    Synthetic peptide derived from the N terminal region of Human Dnmt3b, conjugated to KLH

  • 阳性对照
    • Human testis tissue, HT29 cell lysate, HeLa cells.

性能

  • 形式
    Liquid
  • 存放说明
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term.
  • 存储溶液
    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituents: 48% PBS, 0.88% Sodium chloride, 50% Glycerol
    Note: PBS is without Mg2+, Ca2+
  • Concentration information loading...
  • 纯度
    Immunogen affinity purified
  • 克隆
    多克隆
  • 同种型
    IgG
  • 研究领域

应用

Our Abpromise guarantee covers the use of ab119282 in the following tested applications.

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

应用 Ab评论 说明
WB 1/500 - 1/1000. Predicted molecular weight: 96 kDa.
IHC-P Use a concentration of 10 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF 1/100 - 1/500.

靶标

  • 功能
    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.
  • 组织特异性
    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.
  • 疾病相关
    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.
  • 序列相似性
    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.
  • 结构域
    The PWWP domain is essential for targeting to pericentric heterochromatin.
  • 翻译后修饰
    Sumoylated.
  • 细胞定位
    Nucleus.
  • Information by UniProt
  • 数据库链接
  • 别名
    • 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

图片

  • All lanes : Anti-Dnmt3b antibody (ab119282) at 1/500 dilution

    Lane 1 : HT-29 cell lysate
    Lane 2 : HT-29 cell lysate with blocking peptide

    Predicted band size: 96 kDa

  • ab119282, at 10 µg/ml, staining Dnmt3b in Human testis tissue by immunohistochemistry.
  • ab119282, at 1/100, staining Dnmt3b in HeLa cells by immunofluorescence. The image on the right shows cells treated with blocking peptide.

文献

This product has been referenced in:
  • Ma M  et al. Preeclampsia is associated with hypermethylation of IGF-1 promoter mediated by DNMT1. Am J Transl Res 10:16-39 (2018). IHC-P ; Human . Read more (PubMed: 29422991) »
  • Gailhouste L  et al. Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells. Mol Ther N/A:N/A (2018). WB . Read more (PubMed: 29759938) »
See all 2 Publications for this product

客户评价及客户问答

1-2 of 2 Q&A

Answer

Thank you for your reply.
I am sorry to confirm it is not possible to provide a blocking peptide on this occasion. I apologise for the inconvenience, I understand this would have been useful in this case.

I hope the other suggestions provided will be helpful and look forward to hearing from you with the outcome. Please do not hesitate to let me know if you have any further concerns regarding the results. I hope we can resolve this case for you as soon as possible.

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Question

Inquiry: Antibodies ab4897 lot GR32135-2 and ab119282 lot GR75331-1 (anti Dnmt3a and anti Dnmt3b). I am looking at cytoplasmic and nuclear lysates from HT29 and C4 II cells. I have used an NK-PER kit from Pierce to prepare the lysates, protease inhibitors included. I have run 7.5% SDS PAGE Gels and have blotted onto nitrocellulose membranes. I have used various blocking buffers including TBS/T (+ 5% or 10% Milk Powder), TBS/T + 1%BSA, TBS/T+0.2% fish gelatin, blocking with each at RT and overnight at 4C. I have varied my concentrations of both primary and secondary Abs. I have varied my incubation times for primary Abs and incubation temperatures, over night at 4C or 1 hr to 3hrs at RT. I have used an Anti GAPDH and Anti Dnmt1 both purchased from Abcam at the same time as the Dnmt3a and 3b and have had no problem with those whatsoever. However, with the Anti-Dnmt3a and Anti Dnmt3b I consistantly get multiple strong bands, just about everywhere except at the required molecular weights (I have had faint positivity for Dnmt3b in a HT29 nuclear extract along with multiple other bands). The fact that the Dnmt1 and GAPDH antibodies seem to work very well suggests that the electrophoresis and transfer conditions are OK and the ECL kit is fine. Please could you offer advice as I'm just about running out of ideas? Please could you tell me if there is some information about the nature of the antibodies that you think I am failing to realise? I am using HT29 as a control for Dnmt3b as suggested on your data sheet and our cell line has been verified fairly recently. Is it possible for you to send me an outline of the protocol used to produce the image on the data sheet as my images are not at all similar? Thank you for your help,

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Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from these antibody.

I would like to reassure you that these antibodies are tested and covered by our 6 month guarantee for WB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a creditnote, free of charge replacement or refund.

As requested, I am forwarding a copy of the general WB testing protocols for these antibodies. Please note that these will be a guideline only and may require some further individual optimization by the end user.

ab4897 Anti-Dnmt3a antibody:
A. Preparation of cell lysates
1. Collect cells (confluent T-25) by trypsinization and spin.
2. Lyze the pellet with 100 ul lysis buffer on ice for 10 min. For 500,000 cells, lyze with 20 ul.
3. Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 10 min at 4‹C.
4. Transfer the supernatant to a new tube and discard the pellet.
5. Determine the protein concentration (Bradford assay, A280, or BCA)
6. Take x ul (= y ug protein) and mix with x ul of 2x sample buffer.
7. Boil for 5 min and cool at RT for 5 min.
8. Flash spin to bring down condensation prior to loading gel.
B. Polyacrylamide gel (14.5 cm ˜ 16.5 cm)
1. Agarose plug:
1% agarose dissolved in 1 ˜ Resolving gel buffer.
2. Resolving gel:
24 ml of a 9% gel
5.4 ml 40% acrylamide/bisacrylamide (29:1 mix)
3 ml 8x Resolving gel buffer
15.6 ml water
12 ul TEMED
60 ul 20% ammonium persulfate
3. Stacking gel: 8 ml
1 ml 40% acrylamide/bisacrylamide (29:1 mix)
2 ml 4 ˜ Stacking gel buffer
5 ml water
8 ul TEMED
21.6 ul 20% ammonium persulfate
C. Preparation of gel
1. Assemble the glass plates and spacers (1.5 mm thick).
2. Pour an agarose plug (1-2 mm).
3. Pour the running gel to about 1 cm below the wells of the comb (˜20 ml).
4. Seal with 1 ml water-saturated 1-butanol. (Can stop here and leave gel as is overnight if you want.)
5. When gel has set, pour off the butanol and rinse with deionized water.
6. Pour the stacking gel (˜5 ml) and insert the comb immediately.
7. When the stacking gel has set, place in gel rig and immerse in buffer.
8. Prior to running the gel, flush the wells out thoroughly with running buffer.
D. Running the gel
1. After flash spinning the samples, load into the wells.
2. Be sure to use markers. We use 15 ul Bio-Rad Kaleidoscope Prestained Standards #161-0324 directly.
3. Run with constant current (35-37 mA with voltage set at >150 V).
4. Usual running time is about 1.3 hr.
E. Using precast gels (Ready Gels from Bio-Rad)
1. Assemble gel in gel rig.
2. Prepare protein samples (10 ug will suffice).
3. Use 5 ul of Kaleidoscope standard.
4. Run at 200 V (constant voltage) for 30 min.
F. Preparation of membrane
1. Cut a piece of PVDF membrane.
2. Wet in methanol on a rocker at RT for 5 mins. Remove methanol and add 1x Transfer buffer until ready to use.
G. Membrane transfer
1. Assemble "sandwich":
2. Prewet the sponges, filter papers (slightly bigger than gel) in 1 ˜ Blotting buffer. Sponge - filter paper - gel - membrane - filter paper - sponge
3. Transfer for 1 hr at 15 volts at 4oC on a stir plate. Bigger proteins might take longer to transfer. For a Mini-Transblot, it's 100 V for 1 hr with the cold pack and prechilled buffer.
4. Immerse membrane in Amido-Black stain 5 mins.
5. Destain 4 ˜ 5 mins with destaining buffer.
6. When finished, immerse membrane in Blocking buffer and block for one hour at room temperature.
H. Antibodies and detection
1. Incubate with primary antibody diluted to 2 ug/ml in total volume of 3 mL in Blocking buffer for one hour at room temperature.
2. Wash 4 ˜ 5 min with 0.05% Tween 20 in TBS.
3. Incubate with secondary antibody diluted 1:10,000 (eg HRP anti-rabbit) in Blocking buffer for 1 hour at room temp.
4. Wash 4 ˜ 5 min with 0.05% Tween 20 in TBS.
5. Detect with Chemiluminescent kit.
I. Stripping blot
1. Rinse blot off with 0.05% Tween 20 in PBS.
2. Put blot into Kapak bag cut to slightly bigger size than blot.
3. Add about 5 to 10 ml Stripping buffer.
4. Remove as much air as possible and seal bag.
5. Immerse into 80‹C water bath and incubate for 20 min.
6. Rinse blot off with 0.05% Tween 20 in PBS.
7. Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.
Buffers for Westerns
Lysis buffer:
0.15 M NaCl
5 mM EDTA, pH 8
1% Triton X100
10 mM Tris-Cl, pH 7.4
Just before using add:
1:1000 5 M DTT
1:1000 100 mM PMSF in isopropanol
1:1000 5 M ƒÃ.aminocaproic acid
2x sample buffer:
130 mM Tris-Cl, pH8.0
20% (v/v) Glycerol
4.6% (w/v) SDS
0.02% Bromophenol blue
2% DTT
8x Resolving gel buffer: 100 ml
0.8 g SDS (add last)
36.3 g Trizma base (= 3 M)
Adjust pH to 8.8 with concentrated HCl
4x Stacking gel buffer: 100 ml
0.4 g SDS (add last)
6.05 g Trizma base (= 0.5 M)
Adjust pH to 6.8
10x Running buffer: 1 L
30.3 g Trizma base (= 0.25 M)
144 g Glycine (= 1.92 M)
10 g SDS (= 1%)--add last


ab119282 Anti-Dnmt3b antibody:
Western blot Protocol (Colorimetric)
Blocking
1. Block membrane by incubating 1 hour at room temperature with shaking in Blocking Solution (5% nonfat dry milk, 0.05% Tween-20 in TBS (50mM Tris, 100mM NaCl, pH 7.6).
Incubation with Primary Antibody
1. Dilute primary antibody at the appropriate dilution in Blocking Solution.
2. Incubate the membrane with diluted primary antibody for overnight at 4℃ with agitation.
3. Remove antibody solution. Wash the membrane 3 times for 10 minutes each time at room temperature in TBST (50mM Tris, 100mM NaCl, 0.05% Tween-20, pH 7.6) with shaking. Note: Increase the concentration of Tween-20 to 0.1% reduces the background and increases the specificity, but it will reduce the sensitivity.
Incubation with Second Antibody
1. Incubate membrane with secondary AP conjugate diluted (according to manufacturer’s instructions) in Blocking Solution for 1 hour at room temperature with shaking.
2. Wash the membrane 3 times for 10 minutes each time at room temperature in TBST (50mM Tris, 100mM NaCl, 0.05% Tween-20, pH 7.6) with shaking.
3. Wash membrane with TBS for 2-5 minutes.
Stain
1. Warm up BCIP/NBT Solution (according to manufacturer’s instructions) to room temperature before use.
2. Apply enough of BCIP/NBT solution to cover the specimen completely. Allow 5-10 minutes, under room temperature, for the color to develop.
3. Monitor the color development until desired intensity is observed.
4. Rinse the specimen gently with distilled water to stop the reaction.
Peptide Competition
Before proceeding Western Immunoblotting, add Blocking Peptide to the diluted primary antibody in a molar ratio of 10:1 (peptide to antibody) and incubate the mixture at 4℃ for overnight or at room temperature for 2 hours.
For ab119282, 50 ug of total protein (HT29 cell lysates) was loaded in each lane on an 10% SDS-Page gel. The antibody was used at a concentration of 1 ug/ml diluted in 1% nonfat dry milk, 0.05% Tween-20 in TBS.

I hope these will be helpful to you. However, if you still have some concerns regarding the results I would be pleased to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you are also able to provide images which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.

Order Details
Antibody code:
Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background
Lot number
Purchase order number
or preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
Description of the problem (high background, wrong band size, more bands, no band etc.)
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
Amount of protein loaded

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
Detection method (ECL, ECLPlus etc.)
Positive and negative controls used (please specify)
Optimization attempts (problem solving)
How many times have you tried the Western?
Have you run a "No Primary" control?
Yes No
Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?
What steps have you altered?
Additional Notes:
Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

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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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