使用敲除细胞株进行验证RabMAb

Anti-HDAC2抗体[Y461] (ab32117)

概述

  • 产品名称Anti-HDAC2抗体[Y461]
    参阅全部 HDAC2 一抗
  • 描述
    兔单克隆抗体[Y461] to HDAC2
  • 特异性ab32117 recognises HDAC2.
  • 经测试应用适用于: WB, IHC-P, ICC/IF, Flow Cyt, IP, IHC-Frmore details
  • 种属反应性
    与反应: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide corresponding to residues in the C terminal of human HDAC2.

  • 阳性对照
    • WB: HAP1, HDAC2 knockout HAP1, A431, Hela and K562 cell lysate; Rat brain tissue homogenate. IHC: Human breast carcinoma, Rat spinal cord tissue ICC/IF: MCF-7 cells FC: HeLa cells
  • 常规说明

    This product is a recombinant rabbit monoclonal antibody.

     

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

    Alternative versions available:

    Anti-HDAC2 antibody (Alexa Fluor® 488) [Y461] (ab196471)

    Anti-HDAC2 antibody (Alexa Fluor® 647) [Y461] (ab196518)

    Anti-HDAC2 antibody (HRP) [Y461] (ab195851)

     

性能

应用

Our Abpromise guarantee covers the use of ab32117 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 1/2000. Predicted molecular weight: 55 kDa.
IHC-P Use at an assay dependent concentration.
ICC/IF 1/250 - 1/500.
Flow Cyt 1/60 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP 1/60.
IHC-Fr 1/500. May require antigen retrieval if fixing frozen section in paraformaldehyde.

靶标

  • 功能Responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression and developmental events. Histone deacetylases act via the formation of large multiprotein complexes.
    Forms transcriptional repressor complexes by associating with MAD, SIN3, YY1 and N-COR. Interacts in the late S-phase of DNA-replication with DNMT1 in the other transcriptional repressor complex composed of DNMT1, DMAP1, PCNA, CAF1. Deacetylates TSHZ3 and regulates its transcriptional repressor activity.
  • 组织特异性Widely expressed; lower levels in brain and lung.
  • 序列相似性Belongs to the histone deacetylase family. HD type 1 subfamily.
  • 翻译后修饰S-nitrosylated by GAPDH. In neurons, S-Nitrosylation at Cys-262 and Cys-274 does not affect the enzyme activity but abolishes chromatin-binding, leading to increases acetylation of histones and activate genes that are associated with neuronal development. In embryonic cortical neurons, S-Nitrosylation regulates dendritic growth and branching.
  • 细胞定位Nucleus.
  • Information by UniProt
  • 数据库链接
  • 别名
    • D10Wsu179e antibody
    • HD 2 antibody
    • HD2 antibody
    • HDAC 2 antibody
    • Hdac2 antibody
    • HDAC2_HUMAN antibody
    • Histone deacetylase 2 (HD2) antibody
    • Histone deacetylase 2 antibody
    • OTTHUMP00000017046 antibody
    • OTTHUMP00000227077 antibody
    • OTTHUMP00000227078 antibody
    • RPD3 antibody
    • transcriptional regulator homolog RPD3 antibody
    • YAF1 antibody
    • YY1 associated factor 1 antibody
    • YY1 transcription factor binding protein antibody
    • Yy1bp antibody
    see all

Anti-HDAC2 antibody [Y461] 图像



  • Predicted band size : 55 kDa

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: HDAC2 knockout HAP1 whole cell lysate (20 µg)
    Lane 3: A431 whole cell lysate (20 µg)
    Lane 4: Hela whole cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab32117 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab32117 was shown to specifically react with HDAC2 when HDAC2 knockout samples were used. Wild-type and HDAC2 knockout samples were subjected to SDS-PAGE.  ab32117 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 and 1/10000 respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • ab32117 staining HDAC2 in MCF-7 (human breast carcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain.

    Negative control 1: PBS only.

  • All lanes : Anti-HDAC2 antibody [Y461] (ab32117) at 1/1000 dilution

    Lane 1 : Rat brain tissue homogenate at 20 µg
    Lane 2 : Rat brain tissue homogenate at 40 µg
    Lane 3 : Rat brain tissue homogenate, P1 nuclear fraction at 20 µg
    Lane 4 : Rat brain tissue homogenate, P1 nuclear fraction at 40 µg
    Lane 5 : Rat brain tissue homogenate, P2 non-nuclear fraction at 20 µg
    Lane 6 : Rat brain tissue homogenate, P2 non-nuclear fraction at 40 µg

    Secondary
    HRP-conjugated goat anti-rabbit IgG polyclonal at 1/1000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 55 kDa
    Observed band size : 60 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 35 kDa (possible non-specific binding).

    Exposure time : 5 minutes

    This image is courtesy of an anonymous Abreview

    See Abreview

  • Anti-HDAC2 antibody [Y461] (ab32117) at 1/2000 dilution + K562 cell lysate

    Predicted band size : 55 kDa
    Observed band size : 70 kDa (why is the actual band size different from the predicted?)
  • HDAC2 was immunoprecipitated from 1mg of Hela(Human epithelial cell line from cervix adenocarcinoma)whole cell lysate with ab32117at 1/50 dilution.

    Western blot was performed from the immunoprecipitate using ab32117 at 1/1000 dilution.

    Anti-Rabbit IgG (HRP),specific to the non-reduced form of IgG, was used as secondary antibody at 1/1000 dilution.

    Lane 1: Hela whole cell lysate

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  • Immunohistochemical analysis of HDAC2 expression in paraffin embedded human breast carcinoma tissue section, using 1/250 ab32117.
  • Overlay histogram showing HeLa cells stained with ab32117 (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 (ab32117, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • ab32117 staining HDAC2 in Rat spinal cord tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde and blocked with 1% BSA for 30 minutes at 25°C. Samples were incubated with primary antibody (1/500 in PBS + 0.2% TritonX + 1% BSA) for 16 hours at 4°C. An Alexa Fluor®488-conjugated Donkey anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody. Antigen unmasking with sodium citrate buffer (10mM sodium citrate, 0.05% Tween 20, ph6) was necessary to obtain a good signal. The sections were counterstained with DAPI.

    See Abreview

Anti-HDAC2 antibody [Y461] (ab32117)参考文献

This product has been referenced in:
  • Maiarù M  et al. Complex regulation of the regulator of synaptic plasticity histone deacetylase 2 in the rodent dorsal horn after peripheral injury. J Neurochem 138:222-32 (2016). Read more (PubMed: 26998823) »
  • Soetkamp D  et al. S-nitrosation of mitochondrial connexin 43 regulates mitochondrial function. Basic Res Cardiol 109:433 (2014). Read more (PubMed: 25115184) »

See all 18 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (esophagus)
Gel Running Conditions Non-reduced Non-Denaturing (Native)
Loading amount 40 µg
Specification esophagus
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
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提交于 Sep 05 2016

Application Western blot
Sample Mouse Tissue lysate - whole (lung homogenates)
Gel Running Conditions Reduced Denaturing
Loading amount 25 µg
Specification lung homogenates
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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提交于 Jun 25 2015

Application Western blot
Sample Human Cell lysate - whole cell (Peridontal ligament fibroblast)
Gel Running Conditions Reduced Denaturing (7.5%)
Loading amount 20 µg
Specification Peridontal ligament fibroblast
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
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提交于 Jun 15 2015

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (4-12% gel)
Sample Rat Tissue lysate - nuclear (Brain tissue homogenate, P1 and P2 fractions)
Specification Brain tissue homogenate, P1 and P2 fractions
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
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提交于 Jan 31 2014

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...

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Thank you for your telephone call this afternoon. I am sorry to hear that the current lot of ab32117 is not working so well for you.

I appreciate the time you have spent on these experiments, and it is regrettable the results have not been s...

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Thank you for your call today.

At this point I haven't found a protocol for using whole blood in Western blot. Some of the resources I've found (linked below) indicate that the high content of hemoglobin and other proteins in whole blood, red...

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Thanks you for your email. I will look forward receiving the results soon.

Let me know if this antibody show multiple bands with normal positive control lysates.

Thank you very much for your email.

I have thoroughly checked the protocol again, which looks fine to me. The nuclear extraction kit also have protease inhibitors initso these should also not be a problem.

As per your feedback the a...

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Thank you very much for your email. I am very sorry to hear that you have been observing multiple bands with this antibody.

I am currently searching the cause of this anomaly in publications. There is one publication which shows similar patte...

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