Invitrogen

Phospho-IKK alpha (Ser176, Ser180) Polyclonal Antibody

Advanced Verification

This Antibody was verified by Cell Treatment to ensure that the antibody binds to the antigen stated. View Details

View all (23) IKK alpha antibodies

Datasheet

Datasheet

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Phospho-IKK alpha (Ser176, Ser180) Antibody in Cell Treatment — Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot of Phospho-IKK alpha (Ser176, Ser180) using Phospho-IKK alpha (Ser176, Ser180) polyclonal antibody (Product # 44-714), shows increased expression of Phospho-IKK alpha (Ser176, Ser180) in Jurkat cells upon treatment with TNF-Alpha. {TM}

Phospho-IKK alpha (Ser176, Ser180) Antibody in Immunoprecipitation (IP) — Peptide Competition and Induction, Immunoprecipitates prepared from lysates of Jurkat cells stimulated with TNF-alpha (1-4, 6) or left unstimulated (5) were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% BSA-TBST buffer overnight at 4°C and incubated with 1:500 dilution of Phospho-IKK alpha (Ser176, Ser180) antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5, 6), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphoserine containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F (ab')2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and bands were detected using the Pierce SuperSignal™ method. The data show that the peptide corresponding to Phospho-IKK alpha (Ser176, Ser180) blocks the antibody signal, thereby demonstrating the specificity of the antibody. The data also show that the phospho-signal is induced by the addition of TNF-alpha.

Phospho-IKK alpha (Ser176, Ser180) Antibody in ChIP assay (ChIP) — ChIP- qPCR analysis of IKK-alpha pSer176/pSer180 was performed with 10 µL of the Phospho IKK-alpha pSer176/pSer180 Rabbit polyclonal antibody (Product # 44-714) on sheared chromatin from 2 million HeLa cells treated with 50 ng/mL of TNF Alpha for one hour using the MAGnify™ Chromatin Immunoprecipitation System (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA from each ChIP sample was analyzed by StepOnePlus™ Real-Time PCR System (Product # 4376600) with primers for the promoter of active IL-8 gene, used as positive control target, and the SAT2, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Phospho-IKK alpha (Ser176, Ser180) Antibody in Immunofluorescence (IF) — Immunofluorescence analysis of Phospho-IKK alpha (Ser176, Ser180) was done on 70% confluent log phase HeLa cells. The cells were treated for 20 minutes with 50 ng/mL of TNF-Alpha and fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-IKK alpha (Ser176, Ser180) Rabbit Polyclonal Antibody (Product # 44-714) at 1:250 dilution in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Product Details

Tested Applications

Dilution

ChIP assay (ChIP)

10 µL

Immunocytochemistry (ICC)

1:250

Immunofluorescence (IF)

1:250

Western Blot (WB)

1:250

Product Specifications

Species Reactivity

Human

Host / Isotype

Rabbit / IgG

Class

Polyclonal

Type

Antibody

Immunogen

The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human IKK-alpha that contains serine 176 and serine 180.

Conjugate

Unconjugated

Form

Liquid

Purification

purified

Storage buffer

Dulbecco's PBS, pH 7.3, with 1mg/mL BSA

Contains

0.05% sodium azide

Storage conditions

-20°C

RRID

AB_2533730

Target Information

Nuclear factor kappa B (NF-kappa-B) is a ubiquitous transcription factor and an essential mediator of gene expression during activation of immune and inflammatory responses. NF-kappa-B mediates the expression of a great variety of genes in response to extracellular stimuli including IL-1, TNF-a and bacteria product LPS. NF-kappa-B is associated with Ikappa-B proteins in the cell cytoplasm, which inhibit NF-kappa-B activity. The long-sought Ikappa-B kinase (IKK), which phosphorylates Ikappa-B, and mediates Ikappa-B degradation and NF-kappa-B activation, was recently identified by several laboratories. IKK is a serine protein kinase, and the IKK complex contains alpha and beta subunits (IKK-a and IKK-b). IKK-a and IKK-b interact with each other and both are essential for the NF-kappa-B activation. IKK-a specifically phosphorylates Ikappa-B-alpha. IKK-a is expressed in a variety of human tissues.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Bioinformatics

Protein Aliases: Conserved helix-loop-helix ubiquitous kinase; FLJ40509; I-kappa-B kinase 1; I-kappa-B kinase alpha; I-kappa-B kinase-alpha; IkappaB kinase; IkB kinase alpha subunit; IkBKA; IKBKB; IKK-A; IKK-a kinase; IKK-alpha; IKK1; IKKB; inhibitor of kappa light polypeptide gene enhancer in B-cells; Inhibitor of nuclear factor kappa-B kinase subunit alpha; kinase beta; MGC131801; NFKBIKA; Nuclear facto; Nuclear factor NF-kappa-B inhibitor kinase alpha; Nuclear factor NFkappaB inhibitor kinase alpha; TCF-16; Transcription factor 16

Gene Aliases: CHUK; IKBKA; IKK-alpha; IKK1; IKKA; NFKBIKA; TCF16

UniProt ID: (Human) O15111

Entrez Gene ID: (Human) 1147

Molecular Function: kinase non-receptor serine/threonine protein kinase protein kinase transferase