The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/10000. Detects a band of approximately 85 kDa (predicted molecular weight: 87 kDa).Can be blocked with IKK beta peptide (ab154148).
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. (Heat to 98°C, allow to cool for 10-20 minutes)
Use at an assay dependent concentration.
1/50 - 1/100.
Application notesIs unsuitable for IP.
FunctionActs as part of the IKK complex in the conventional pathway of NF-kappa-B activation and phosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappa-B complex and ultimately the degradation of the inhibitor. Also phosphorylates NCOA3.
Tissue specificityHighly expressed in heart, placenta, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis and peripheral blood.
Sequence similaritiesBelongs to the protein kinase superfamily. Ser/Thr protein kinase family. I-kappa-B kinase subfamily. Contains 1 protein kinase domain.
Post-translational modificationsUpon cytokine stimulation, phosphorylated on Ser-177 and Ser-181 by MEKK1 and/or MAP3K14/NIK; which enhances activity. Once activated, autophosphorylates on the C-terminal serine cluster; which decreases activity and prevents prolonged activation of the inflammatory response. Acetylation of Thr-180 by Yersinia yopJ prevents phosphorylation and activation, thus blocking the I-kappa-B pathway. Ubiquitinated. Monoubiquitination involves TRIM21 that leads to inhibition of Tax-induced NF-kappa-B signaling. According to PubMed:19675099, 'Ser-163' does not serve as a monoubiquitination site. According to PubMed:16267042, ubiquitination on 'Ser-163' modulates phosphorylation on C-terminal serine residues. Monoubiquitination by TRIM21 is dirupted by Yersinia yopJ.
Cellular localizationCytoplasm. Membrane raft. Colocalized with DPP4 in membrane rafts.
Western blot - Anti-IKK beta antibody [EPR6043] (ab124957)
Predicted band size : 87 kDa
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: IKK beta knockout HAP1 cell lysate (20 µg) Lanes 1 and 2: Merged signal (red and green). Green - ab124957 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa. ab124957 was shown to specifically react with IKK beta when IKK beta knockout samples were used. Wild-type and IKK beta knockout samples were subjected to SDS-PAGE. ab124957 and ab8245 (loading control to GAPDH) were diluted 1/500 and 1/2000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.
Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling IKK beta with unpurified ab124957 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised 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.