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

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Datasheet

Questions & Answers

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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.

Phospho-IKK alpha (Ser176, Ser180) Antibody in Western Blot (WB) — Peptide Competition and Induction, Immunoprecipitates prepared from lysates of Jurkat cells stimulated with 80 ng/mL TNF-alpha for 5 minutes at 37C (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 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}

Product Details

Applications

Tested Dilution

Publications

ChIP assay (ChIP)

10 µL

Immunocytochemistry (ICC)

1:250

Immunofluorescence (IF)

1:250

Western Blot (WB)

1:1000

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

Inhibitor of Nuclear factor Kappa-B kinase subunit alpha (IKK alpha) is encoded by the CHUK gene and located on chromosome 10 in humans. It is also known as IKK1 or CHUK (Conserved helix-loop-helix ubiquitous kinase). IKK alpha belongs to the family of serine threonine protein kinase and forms a part of IKK complex. The IKK complex is required for the activation of NF-kB with IKK alpha specifically known to be involved in the non-canocial pathway for NF-kB activation. IKK alpha along with IKK beta and a regulatory protein NEMO (IKK gamma) phosphorylates IkB proteins which targets the later for ubiquitin mediated degradation. It is involved with several cellular processes with extensive studies on its role in inflammation and cell survival.

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

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Bioinformatics

Protein Aliases: Conserved helix-loop-helix ubiquitous kinase; EC 2.7.11.10; FLJ40509; I-kappa-B kinase 1; I-kappa-B kinase alpha; I-kappa-B kinase-alpha; ikappaB kinase; IkB kinase alpha subunit; IKBKB; IKK-a kinase; IKK-B; IKK-beta; IKK1; IKKB; Inhibitor of nuclear factor kappa-B kinase subunit alpha; kinase beta; MGC131801; NFKBIKA; 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