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,IHC-P,IHC-Fr,IF,FCM

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