Overview

  • Product name
    Anti-IKB alpha antibody [E130]
    See all IKB alpha primary antibodies
  • Description
    Rabbit monoclonal [E130] to IKB alpha
  • Host species
    Rabbit
  • Specificity
    This antibody detects both the phosphorylated and non-phosphorylated form of the serine 32 region of IKB alpha.
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-P, IP, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Hamster, Human
    Predicted to work with: Cow, Pig
  • Immunogen

    Synthetic peptide within Human IKB alpha. The exact sequence is proprietary.
    Database link: P25963

  • Positive control
    • WB: Wild type HAP1 whole cell lysate. HeLa whole cell lysate. PC-12, SH-SY5Y, HepG2, K562, NIH/3T3 and RAW 264.7 cell lysate. Human fetal liver lysate. IHC-P: Human prostate carcinoma and stomach tissue. Rat kidney tissue. ICC/IF: HeLa and RAW 264.7 cells. IP: IKB alpha IP in HeLa cell lysate. Flow Cyt: HeLa cells.
  • General notes

      

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab32518 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 1/50.
WB 1/1000 - 1/100000. Detects a band of approximately 35 kDa (predicted molecular weight: 36 kDa).
IHC-P 1/100.
IP 1/20.
Flow Cyt 1/20.

Target

  • Function
    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.
  • Involvement in disease
    Ectodermal dysplasia, anhidrotic, with T-cell immunodeficiency autosomal dominant
  • Sequence similarities
    Belongs to the NF-kappa-B inhibitor family.
    Contains 5 ANK repeats.
  • Post-translational
    modifications
    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.
  • Cellular localization
    Cytoplasm. Nucleus. Shuttles between the nucleus and the cytoplasm by a nuclear localization signal (NLS) and a CRM1-dependent nuclear export.
  • Information by UniProt
  • Database links
  • Alternative names
    • 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

Images

  • 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)

    Lanes 1 - 3: Merged signal (red and green). Green - ab32518 observed at 38 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab32518 was shown to specifically react with IKB alpha in wild-type HAP1 cells. No band was observed when IKB alpha knockout samples were tested. Wild-type and IKB alpha knockout samples were subjected to SDS-PAGE. ab32518 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1/10,000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

  • Immunohistochemical staining of paraffin embedded human stomach with purified ab32518 at a working dilution of 1 in 100.

    The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

    PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

  • Unpurified ab32518 staining IKB alpha in RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells treated with FK506 (ab120223), by ICC/IF. Decrease in IkBα/&beta expression correlates with increased concentration of FK506, as described in literature.
    The cells were incubated at 37°C for 3h in media containing different concentrations of ab120223 (FK506) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature.

    Staining of the treated cells with ab32518 (1/100 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight® 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • Immunofluorescence staining of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells with purified ab32518 at a working dilution of 1 in 50, counterstained with DAPI.

    The secondary antibody was ab150077, Alexa Fluor® 488 goat anti rabbit, used at a dilution of 1 in 500. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100.

    The negative control is shown in bottom right hand panel - for the negative control, purified ab32518 was used at a dilution of 1/50 followed by ab150120, Alexa Fluor® 594 goat anti-mouse antibody at a dilution of 1/500.

  • ab32518 (purified) at 1/20 immunoprecipitating IKB alpha in HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate (Lane 1). For western blotting a HRP-conjugated goat anti-rabbit IgG was used as the secondary antibody (1/1000).

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

  • All lanes : Anti-IKB alpha antibody [E130] (ab32518) at 1/10000 dilution (purified)

    Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma cell line) cell lysate
    Lane 2 : NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate
    Lane 3 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 36 kDa
    Observed band size: 35 kDa
    why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using unpurified ab32518 at 1/50 dilution.

  • Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling IKB alpha with purified ab32518 at 1/20 dilution (10 µg/mL) (red).

    Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

  • All lanes : Anti-IKB alpha antibody [E130] (ab32518) at 1/10000 dilution (purified)

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
    Lane 2 : K562 Human chronic myelogenous leukemia cell line from bone marrow) cell lysate
    Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 36 kDa
    Observed band size: 35 kDa why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunohistochemical staining of paraffin embedded rat kidney with purified ab32518 at a working dilution of 1 in 100.

    The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

    PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

  • ICC/IF image of unpurified ab32518 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

    The cells were fixed in 100% methanol (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab32518, 1/1000 dilution) 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 1.43 µM.

  • Anti-IKB alpha antibody [E130] (ab32518) at 1/1000 dilution (purified) + Human fetal liver at 10 µg

    Secondary
    HRP anti-rabbit IgG, specific to the non reduced form of IgG at 1/1000 dilution

    Predicted band size: 36 kDa
    Observed band size: 35 kDa why is the actual band size different from the predicted?



    Blocking/Dilution buffer: 5% NFDM/TBST.

  • Anti-IKB alpha antibody [E130] (ab32518) at 1/1000 dilution (purified) + SH-SY5Y cell lysate at 10 µg

    Secondary
    HRP goat anti-rabbit (H+L) at 1/1000 dilution

    Predicted band size: 36 kDa
    Observed band size: 35 kDa why is the actual band size different from the predicted?



    Blocking buffer: 5% NFDM/TBST

    Dilution buffer: 5% NFDM/TBST

  • Anti-IKB alpha antibody [E130] (ab32518) at 1/10000 dilution (unpurified) + HeLa cell lysate

    Predicted band size: 36 kDa
    Observed band size: 35 kDa why is the actual band size different from the predicted?

  • Unpurified ab32518 used to immunoprecipitate IKB alpha from HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate. The antibody was further used to Western blot the protein.

    Lane 1: IKB alpha IP

    Lane 2: Control immunoprecipitate

    Lane 3: Input (20%)

    See Abreview

  • Unpurified ab32518 used to immunoprecipitate IKB alpha from PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate. The antibody was further used to Western blot the protein.

    Lane 1: IKB alpha IP

    Lane 2: Control immunoprecipitate

    Lane 3: Input (20%)

    See Abreview

  • Unpurified ab32518 at 1/100 staining mouse kidney tissue sections by IHC-P.

    The tissue was paraformaldehyde fixed and a heat mediated antigen retrieval step in citrate buffer was performed before the tissue was blocked and incubated with the antibody for 1 hour. An HRP conjugated goat anti-rabbit antibody was used as the secondary.

    See Abreview

References

This product has been referenced in:
  • Chen Y  et al. Epigenetically upregulated oncoprotein PLCE1 drives esophageal carcinoma angiogenesis and proliferation via activating the PI-PLCe-NF-?B signaling pathway and VEGF-C/ Bcl-2 expression. Mol Cancer 18:1 (2019). Read more (PubMed: 30609930) »
  • Dai C  et al. Baicalin protects human retinal pigment epithelial cell lines against high glucose-induced cell injury by up-regulation of microRNA-145. Exp Mol Pathol 106:123-130 (2019). Read more (PubMed: 30625293) »
See all 134 Publications for this product

Customer reviews and Q&As

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1-10 of 29 Abreviews

Application
Western blot
Sample
Mouse Cell lysate - whole cell (Endothelial)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
Endothelial
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jul 28 2017

Application
Western blot
Sample
Rat Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
20 µg
Specification
Skeletal muscle
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 18 2016

Application
Western blot
Sample
Mouse Tissue lysate - whole (Kidney)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Treatment
28 min IRI
Specification
Kidney
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Dr. Wesley Konsavage

Verified customer

Submitted Sep 15 2015

Application
Western blot
Sample
Human Cell lysate - whole cell (human head and neck cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Treatment
10 nM PMA
Specification
human head and neck cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Aug 31 2015

Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing (12% gel)
Sample
Mouse Tissue lysate - whole (Skeletal muscle (AT))
Specification
Skeletal muscle (AT)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 27 2014

Abreviews
Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing (12%)
Sample
Rat Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jun 17 2013

Abreviews
Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing (12%)
Sample
Mouse Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jun 17 2013

Abreviews
Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Reduced Denaturing (12%)
Sample
Human Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jun 17 2013

Application
Western blot
Sample
Human Cell lysate - whole cell (smooth muscle cells)
Loading amount
25 µg
Specification
smooth muscle cells
Treatment
infection with interested gene for 48 hours
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 16 2013

Application
Western blot
Sample
Rat Cell lysate - whole cell (Hepatocytes)
Loading amount
50 µg
Specification
Hepatocytes
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Dec 06 2012

1-10 of 29 Abreviews

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