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Blank control (blue line): MCF 7 (blue). Primary Antibody (green line): Rabbit Anti-SIRT1 antibody (orb11372), Dilution: 3 µg/10 5 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 70% methanol (Overnight at 4C) and then permeabilized with 90% ice-cold methanol for 30 min on ice. Cells stained with Primary Antibody for 30 min at room temperature. The cells were then incubated in 1 X 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-SIRT1 antibody (orb11372), Dilution: 1 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 0.1% PBST for 20 min at room temperature. 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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Blank control: HL-60. Primary Antibody (green line): Rabbit Anti-SIRT1 antibody, Dilution: 1 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 0.1% PBST for 20 min at room temperature. 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 (human glioma tissue), 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 (SIRT1) Polyclonal Antibody, Unconjugated (orb11372) 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 (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 37C for 30 min, Antibody incubation with (SIRT1) Polyclonal Antibody, Unconjugated (orb11372) at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining. |
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Sample: Hela (Human) Cell Lysate at 30 ug, Hela KO SIRT1 (Human) Cell Lysate at 30 ug, Primary: Anti-SIRT1 (orb11372) at 1/500 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 58/81 kD, Observed band size: 75/150 kD. |
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Sample: Lane 1: Hela (Human) Cell Lysate at 30 ug, Lane 2: NIH/3T3 (Mouse) Cell Lysate at 30 ug, Lane 3: HepG2 (Human) Cell Lysate at 30 ug, Lane 4: 293T (Human) Cell Lysate at 30 ug, Lane 5: Testis (Mouse) Lysate at 40 ug, Primary: Anti-SIRT1 (orb11372) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 116/95 kD, Observed band size: 116 kD. |
