1/100 - 1/250. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Acts 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. As part of the non-canonical pathway of NF-kappa-B activation, the MAP3K14-activated CHUK/IKKA homodimer phosphorylates NFKB2/p100 associated with RelB, inducing its proteolytic processing to NFKB2/p52 and the formation of NF-kappa-B RelB-p52 complexes. Also phosphorylates NCOA3. Phosphorylates 'Ser-10' of histone H3 at NF-kappa-B-regulated promoters during inflammatory responses triggered by cytokines.
Defects in CHUK are the cause of cocoon syndrome (COCOS) [MIM:613630]; also known as fetal encasement syndrome. COCOS is a lethal syndrome characterized by multiple fetal malformations including defective face and seemingly absent limbs, which are bound to the trunk and encased under the skin.
Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. I-kappa-B kinase subfamily. Contains 1 protein kinase domain.
Phosphorylated by MAP3K14/NIK, AKT and to a lesser extent by MEKK1, and dephosphorylated by PP2A. Autophosphorylated. Acetylation of Thr-179 by Yersinia yopJ prevents phosphorylation and activation, thus blocking the I-kappa-B signaling pathway.
Cytoplasm. Nucleus. Shuttles between the cytoplasm and the nucleus.
Nuclear Factor Of Kappa Light Chain Gene Enhancer In B Cells Inhibitor antibody
Transcription factor 16 antibody
Western blot - Anti-IKK alpha antibody [EPR464] (ab109749)
Lanes 1, 3 and 5:Wild-type HAP1 cell lysate (20 µg) Lanes 2, 4 and 6: IKK alpha knockout HAP1 cell lysate (20 µg) Lanes 1 and 2: Green signal from target - ab109749 observed at 84 kDa Lanes 3 and 4: Red signal from loading control - ab8226 observed at 42 kDa Lanes 5 and 6: Merged (red and green) signal ab109749 was shown to specifically react with IKK alpha when IKK alpha knockout samples were used. Wild-type and IKK alpha knockout samples were subjected to SDS-PAGE. ab109749 and ab8226 (loading control to beta actin) were both diluted at 1/1000 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 1/10000 dilution for 1 h at room temperature before imaging.
Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling IKK alpha with purified ab109749 at 1/250 dilution. Cells were fixed with 4% PFA and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG (Alexa Fluor®488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.
Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling IKK alpha with unpurified ab109749 at 1/50 dilutio n(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.
Western blot - IKK alpha antibody [EPR464] (ab109749)
All lanes : Anti-IKK alpha antibody [EPR464] (ab109749) at 1/1000 dilution
Lane 1 : Jurkat cell lysate Lane 2 : HeLa cell lysate Lane 3 : HepG2 cell lysate Lane 4 : Daudi cell lysate