使用敲除细胞株进行验证

Anti-IKB alpha抗体[6A920] (ab12134)

概述

  • 产品名称
    Anti-IKB alpha抗体[6A920]
    参阅全部 IKB alpha 一抗
  • 描述
    小鼠单克隆抗体[6A920] to IKB alpha
  • 特异性
    Ab12134 recognises IKB alpha.
  • 经测试应用
    适用于: IHC-P, WBmore details
  • 种属反应性
    与反应: Mouse, Rat, Hamster, Human, African green monkey
  • 免疫原

    6xHis-tagged fusion protein, corresponding to amino acids 105-291 of Human IKB alpha.

  • 阳性对照
    • Jurkat + PMA (10 nm/ml) + ionomycin (1 µM) cell extract.

性能

应用

Our Abpromise guarantee covers the use of ab12134 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
IHC-P Use at an assay dependent concentration.
WB Use a concentration of 1 - 2 µg/ml. Detects a band of approximately 40 kDa (predicted molecular weight: 35.6 kDa).

靶标

  • 功能
    Inhibits the activity of dimeric NF-kappa-B/REL complexes by trapping REL dimers in the cytoplasm through masking of their nuclear localization signals. On cellular stimulation by immune and proinflammatory responses, becomes phosphorylated promoting ubiquitination and degradation, enabling the dimeric RELA to translocate to the nucleus and activate transcription.
  • 疾病相关
    Ectodermal dysplasia, anhidrotic, with T-cell immunodeficiency autosomal dominant
  • 序列相似性
    Belongs to the NF-kappa-B inhibitor family.
    Contains 5 ANK repeats.
  • 翻译后修饰
    Phosphorylated; disables inhibition of NF-kappa-B DNA-binding activity. Phosphorylation at positions 32 and 36 is prerequisite to recognition by UBE2D3 leading to polyubiquitination and subsequent degradation.
    Sumoylated; sumoylation requires the presence of the nuclear import signal. Sumoylation blocks ubiquitination and proteasome-mediated degradation of the protein thereby increasing the protein stability.
    Monoubiquitinated at Lys-21 and/or Lys-22 by UBE2D3. Ubiquitin chain elongation is then performed by CDC34 in cooperation with the SCF(FBXW11) E3 ligase complex, building ubiquitin chains from the UBE2D3-primed NFKBIA-linked ubiquitin. The resulting polyubiquitination leads to protein degradation. Also ubiquitinated by SCF(BTRC) following stimulus-dependent phosphorylation at Ser-32 and Ser-36.
    Deubiquitinated by porcine reproductive and respiratory syndrome virus Nsp2 protein, which thereby interferes with NFKBIA degradation and impairs subsequent NF-kappa-B activation.
  • 细胞定位
    Cytoplasm. Nucleus. Shuttles between the nucleus and the cytoplasm by a nuclear localization signal (NLS) and a CRM1-dependent nuclear export.
  • Information by UniProt
  • 数据库链接
  • 别名
    • I kappa B alpha antibody
    • I-kappa-B-alpha antibody
    • IkappaBalpha antibody
    • IkB-alpha antibody
    • IKBA antibody
    • IKBA_HUMAN antibody
    • IKBalpha antibody
    • MAD 3 antibody
    • MAD3 antibody
    • Major histocompatibility complex enhancer-binding protein MAD3 antibody
    • NF kappa B inhibitor alpha antibody
    • NF-kappa-B inhibitor alpha antibody
    • NFKBI antibody
    • NFKBIA antibody
    • Nuclear factor of kappa light chain gene enhancer in B cells antibody
    • Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha antibody
    see all

Anti-IKB alpha antibody [6A920] 图像



  • Predicted band size : 35.6 kDa

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: IKB alpha knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)
    Lane 4: HepG2 whole cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab12134 observed at 38 kDa. Red - loading control, ab176560, observed at 50 kDa.

    ab12134 was shown to specifically react with IKB alpha in wild type cells as signal was lost in IKB alpha knockout cells. Wild-type and IKB alpha knockout samples were subjected to SDS-PAGE. Ab12134 and ab176560 (Rabbit anti alpha Tubulin loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  • All lanes : Anti-IKB alpha antibody [6A920] (ab12134) at 1/1000 dilution

    Lane 1 : Human B cell lysate treated with CD40L for 4 hours
    Lane 2 : Human B cell lysate treated with CD40L for 4 hours
    Lane 3 : Human B cell lysate treated with CD40L for 4 hours

    Lysates/proteins at 10 µg per lane.

    Secondary
    HRP-conjugated sheep anti-mouse IgG polyclonal at 1/5000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 35.6 kDa
    Observed band size : 37 kDa (why is the actual band size different from the predicted?)


    Exposure time : 10 minutes

    This image is courtesy of an anonymous Abreview

    See Abreview

  • ab12134 at 1/250 staining human adrenal tissue sections by IHC-P. The tissue was formaldehyde fixed and a heat mediated antigen retrieval step in citrate buffer was performed. The tissue was blocked in serum before incubation with the primary antibody for 30 minutes at 22°C. An HRP conjugated goat anti-mouse antibody was used as the secondary.

    See Abreview



  • Predicted band size : 35.6 kDa

    ab12134 detects both phosphorylated and non-phosphorylated forms of IKB alpha by Western blot. Jurkat cells (1E7) were treated for indicated time periods with 10 nm/ml PMA, 1 µM ionomycin and ab12134 (1/10000 dilution). 10 µl of cytoplasmic (C) and nuclear extract (N) were resolved on a 8.75% SDS-PAGE and transferred to Immobilon membrane.

    ab12134 detects both phosphorylated and non-phosphorylated forms of IKB alpha by Western blot. Jurkat cells (1E7) were treated for indicated time periods with 10 nm/ml PMA, 1 µM ionomycin and ab12134 (1/10000 dilution). 10 µl of cytoplasmic (C) and nuclear extract (N) were resolved on a 8.75% SDS-PAGE and transferred to Immobilon membrane.

Anti-IKB alpha antibody [6A920] (ab12134)参考文献

This product has been referenced in:
  • Chang MS  et al. Epstein-Barr virus-encoded BARF1 promotes proliferation of gastric carcinoma cells through regulation of NF-?B. J Virol 87:10515-23 (2013). WB ; Human . Read more (PubMed: 23824821) »

See 1 Publication for this product

Product Wall

Application
Western blot
Loading amount
10 µg
Gel Running Conditions
Reduced Denaturing (10)
Sample
Human Cell lysate - whole cell (B cells)
Specification
B cells
Treatment
CD40L for 4 hours
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

提交于 Oct 27 2014

Thank you for submitting an Abreview for ab12134. As you may have noticed, your review has now been published on our website.
Since you obtained poor results using the antibody in a untested species/application, we would like to follow up on this ...

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cultured Cells (RAW 264.7)
Specification
RAW 264.7
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton-X100 in 2% BSA for 15min
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 4°C
Username

Dr. Mahesh Shivananjappa

Verified customer

提交于 Jun 21 2012

Abcam has not validated the combination of species/application used in this Abreview.
Application
Flow Cytometry
Sample
Human Cell (Monocytes from Apheresis)
Specification
Monocytes from Apheresis
Fixation
BD Cytofix/Cytoperm 20 min
Permeabilization
Yes - BD Cytofix/Cytoperm 20 min
Gating Strategy
Monocytes
Username

Dr. Mahesh Shivananjappa

Verified customer

提交于 Dec 08 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Platelets)
Loading amount
20 µg
Specification
Platelets
Treatment
A23187 for 1 h
Gel Running Conditions
Reduced Denaturing (4-20%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Dr. Mahesh Shivananjappa

Verified customer

提交于 Nov 14 2011

Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (U2OS cell line)
Total protein in input
500 µg
Specification
U2OS cell line
Immuno-precipitation step
Protein G
Username

Abcam user community

Verified customer

提交于 Oct 30 2007

Application
Immunoprecipitation
Sample
Mouse Cell lysate - whole cell (3T3 cell line)
Total protein in input
500 µg
Specification
3T3 cell line
Immuno-precipitation step
Protein G
Username

Abcam user community

Verified customer

提交于 Oct 30 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Cell lysate - whole cell (PC12)
Loading amount
20 µg
Specification
PC12
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
Username

Abcam user community

Verified customer

提交于 Oct 09 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (adrenal)
Specification
adrenal
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer pH 6.0
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 22°C
Username

Abcam user community

Verified customer

提交于 Oct 05 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (adrenal)
Specification
adrenal
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer pH 6.0
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 22°C
Username

Abcam user community

Verified customer

提交于 Oct 05 2007

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