Anti-Phospho-IKK beta (Ser466) Polyclonal Antibody is a Rabbit antibody targeting Phospho-IKK beta (Ser466). Anti-Phospho-IKK beta (Ser466) Polyclonal Antibody can be used in IHC-P,IHC-Fr,ICC/IF,IF,FCM.

IgG

Human,Mouse,Rat (predicted:Dog,Pig,Cow,Rabbit)

1. Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01 M, pH 6.0), Boiling bathing for 15 min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min; Blocking buffer (normal goat serum) at 37℃ for 20 min;
Incubation: Anti-phospho-IKK beta (Ser466) Polyclonal Antibody, Unconjugated (TMAB-10846) 1: 200, overnight at 4°C, followed by conjugation to the secondary antibody and DAB staining
2. Paraformaldehyde-fixed, paraffin embedded (Mouse brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-IKK betA (Ser466)) Polyclonal Antibody, Unconjugated (TMAB-10846) at 1:500 overnight at 4°C, followed by a conjugated secondary for 20 minutes and DAB staining.
3. A549 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (phospho-IKK beta (Ser466)) polyclonal Antibody, Unconjugated (TMAB-10846) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.
4. Blank control: A549.
Primary Antibody (green line): Rabbit Anti-phospho-IKK beta (Ser466) antibody (TMAB-10846)
Dilution: 1 μg/Test;
Secondary Antibody (white blue line) : Goat anti-rabbit IgG-AF488
Dilution: 0.5 μg/Test.
Isotype control (orange line): Normal Rabbit IgG
Protocol
The cells were incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20,000 events was performed.

IHC-P: 1:100-500; IHC-Fr: 1:100-500; ICC/IF: 1:100-500; IF: 1:100-500; FCM: 1μg/Test

Polyclonal

KLH conjugated Synthesised phosphopeptide: human IKK beta around the phosphorylation site of Ser466

Human

IKBKB

Inhibitor of nuclear factor kappa-B kinase subunit beta

Serine kinase that plays an essential role in the NF-kappa-B signaling pathway which is activated by multiple stimuli such as inflammatory cytokines, bacterial or viral products, DNA damages or other cellular stresses. Acts as part of the canonical IKK complex in the conventional pathway of NF-kappa-B activation and phosphorylates inhibitors of NF-kappa-B on 2 critical serine residues. These modifications allow polyubiquitination of the inhibitors and subsequent degradation by the proteasome. In turn, free NF-kappa-B is translocated into the nucleus and activates the transcription of hundreds of genes involved in immune response, growth control, or protection against apoptosis. In addition to the NF-kappa-B inhibitors, phosphorylates several other components of the signaling pathway including NEMO/IKBKG, NF-kappa-B subunits RELA and NFKB1, as well as IKK-related kinases TBK1 and IKBKE. IKK-related kinase phosphorylations may prevent the overproduction of inflammatory mediators since they exert a negative regulation on canonical IKKs. Also phosphorylates other substrates including NCOA3, BCL10 and IRS1. Within the nucleus, acts as an adapter protein for NFKBIA degradation in UV-induced NF-kappa-B activation.