Anti-Phospho-IkB alpha/NFKBIA (Tyr305) Polyclonal Antibody is a Rabbit antibody targeting Phospho-IkB alpha/NFKBIA (Tyr305). Anti-Phospho-IkB alpha/NFKBIA (Tyr305) Polyclonal Antibody can be used in WB,IHC-P,IHC-Fr,IF,FCM.

IgG

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

1. 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 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (phospho-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (TMAB-01428) at 1:400 overnight at 4°C, followed by a conjugated secondary antibody for 20 min and DAB staining.
2. Paraformaldehyde-fixed, paraffin embedded (Mouse liver); 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 (p-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (TMAB-01428) at 1:400 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
3. Tissue/cell: human colon carcinoma; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01M, pH6.0), Boiling bathing for 15 min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min; Blocking buffer (normal goat serum) at 37°C for 20 min;
Incubation: Anti-NFKBIA Polyclonal Antibody, Unconjugated (TMAB-01428) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
4. Blank control (blue line): Jurkat (fixed with 2% paraformaldehyde (10 min), then permeabilized with 90% ice-cold methanol for 30 min on ice).
Primary Antibody (green line): Rabbit Anti-phospho-IKB alpha (Tyr305) antibody (TMAB-01428), Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC, Dilution: 1 μg/test.
5. Sample: 293T Cell (Human) Lysate at 40 μg
Primary: Anti-p-NFKBIa (Tyr305)(TMAB-01428) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 35 kDa
Observed band size: 38 kDa
6. Blank control (blue line): Hela (fixed with 70% methanol (Overnight at 4°C) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C).
Primary Antibody (green line): Rabbit Anti-phospho-IKB alpha (Tyr305) antibody (TMAB-01428), Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-PE, Dilution: 1 μg/test.
7. Blank control: Hela.
Primary Antibody (green line): Rabbit Anti-phospho-IKB alpha (Tyr305) antibody (TMAB-01428)
Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody: Goat anti-rabbit IgG-AF647
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.
8. Paraformaldehyde-fixed, paraffin embedded (rat lymphoid); 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-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (TMAB-01428) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
9. Paraformaldehyde-fixed, paraffin embedded (rat stomach); 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-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (TMAB-01428) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
10. Paraformaldehyde-fixed, paraffin embedded (rat adrenal gland); 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-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (TMAB-01428) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
11. Paraformaldehyde-fixed, paraffin embedded (rat stomach); 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-IKB alpha (Tyr305)) Polyclonal Antibody, Unconjugated (TMAB-01428) at 1:200 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: 1μg/Test

Polyclonal

KLH conjugated Synthesised phosphopeptide: human IKB alpha around the phosphorylation site of Tyr305

Human

NFkB pathway,NFkB pathway,TLR Signaling,NFkB Pathway