Anti-Phospho-NF-kB p65 (Thr254) Polyclonal Antibody is a Rabbit antibody targeting Phospho-NF-kB p65 (Thr254). Anti-Phospho-NF-kB p65 (Thr254) Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,IF,FCM.

IgG

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

1. Paraformaldehyde-fixed, paraffin embedded (Rat colon); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-NFKB p65 (Thr254)) Polyclonal Antibody, Unconjugated (TMAB-01468) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
2. Sample:
Lung (Mouse) Lysate at 40 μg
Spleen (Mouse) Lysate at 40 μg
Primary: Anti-phospho-NFKB p65 (Thr254) (TMAB-01468) at 1/300 dilution
Secondary: HRP conjugated Goat-Anti-rabbit IgG (secondary antibody) at 1/5000 dilution
Predicted band size: 61 kDa
Observed band size: 61 kDa
3. Overlay histogram showing HL 60 cells stained with TMAB-01468 (Green line). The cells were fixed with 90% methanol (5 min) and then permeabilized with 0.01M PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (TMAB-01468,3 μg/1x10^6 cells) for 30 min at 22°C. The secondary antibody used was fluorescein isothiocyanate goat anti-rabbit IgG (H+L) (Brillant blue line) at 1/200 dilution for 30 min at 22°C. Isotype control antibody was rabbit IgG (polyclonal) (3 μg/1x10^6 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of 20,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
4. Blank control: A431. Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Thr254) antibody (TMAB-01468)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-FITC
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C. The cells were then 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.
5. Blank control: Mouse spleen.
Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Thr254) antibody (TMAB-01468)
Dilution: 2 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-AF488
Dilution: 1 μg/test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C. The cells were then 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.
6. Paraformaldehyde-fixed, paraffin embedded (Human cervical carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-NFKB p65 (Thr254)) Polyclonal Antibody, Unconjugated (TMAB-01468) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
7. Paraformaldehyde-fixed, paraffin embedded (Human stomach carcinoma); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-NFKB p65 (Thr254)) Polyclonal Antibody, Unconjugated (TMAB-01468) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; IF: 1:100-500; FCM: 2ug/Test

Polyclonal

KLH conjugated Synthesised phosphopeptide: human NFKBp65 around the phosphorylation site of Thr254

Human

RELA

Transcription factor p65

RelA (p65),NFkB pathway,Inflammatory mediators,RelA (p65),NFkB pathway,Obesity,Host Immune Response,Host Virus Interaction,Alzheimer's disease,NFkB Pathway

NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. NF-kappa-B heterodimeric p65-p50 and p65-c-Rel complexes are transcriptional activators. The NF-kappa-B p65-p65 complex appears to be involved in invasin-mediated activation of IL-8 expression. The inhibitory effect of I-kappa-B upon NF-kappa-B the cytoplasm is exerted primarily through the interaction with p65. p65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1.