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Blank control (blue line): Hela (blue). Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Ser536) antibody (orb6504), 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. Protocol, The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1X PBS/2% BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed. |
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Blank control (blue line): HL60 (blue). Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Ser536) antibody (orb6504), 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. Protocol, The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1X PBS/2% BSA/10% goat serum to block non-specific protein-protein interactions followed by the antibody for 15 min at room temperature. The secondary antibody used for 40 min at room temperature. Acquisition of 20000 events was performed. |
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Blank control: HL-60. Primary Antibody (green line): Rabbit Anti-phospho-NFKB p65 (Ser536) antibody (orb6504), 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 -20C. 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. Acquisition of 20000 events was performed. |
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Paraformaldehyde-fixed, paraffin embedded (mouse bladder), 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 37C for 30 min, Antibody incubation with (phospho-NFKB p65 (Ser536)) Polyclonal Antibody, Unconjugated (orb6504) at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining. |
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Paraformaldehyde-fixed, paraffin embedded (rat spleen), 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 37C for 30 min, Antibody incubation with (phospho-NFKB p65 (Ser536)) Polyclonal Antibody, Unconjugated (orb6504) at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining. |
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Sample: Lane 1: MCF-7 (Human) Cell Lysate at 30 ug, Lane 2: A431 (Human) Cell Lysate at 30 ug, Lane 3: HL60 (Human) Cell Lysate at 30 ug, Lane 4: Raw264.7 (Mouse) Cell Lysate at 30 ug, Lane 5: NIH/3T3 (Mouse) Cell Lysate at 30 ug, Lane 6: Jurkat (Human) Cell Lysate at 30 ug, Lane 7: A549 (Human) Cell Lysate at 30 ug, Primary: Anti-phospho-NFKB p65 (Ser536) (orb6504) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 65 kD, Observed band size: 65 kD. |
