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Western blot analysis of rat brain lysates(Lane1),rat heart lysates(Lane2) using 14-3-3 antibody |
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Blank control (blue line): A549 (blue). Primary Antibody (green line): Rabbit Anti-14-3-3 Alpha + Beta + Gamma + Delta + Epsilon antibody (orb13223), 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 70% ethanol (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 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: A431. Primary Antibody (green line): Rabbit Anti-14-3-3 Alpha + Beta + Gamma + Delta + Epsilon antibody (orb13223), 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 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 (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 (14-3-3 Alpha + Beta + Gamma + Delta + Epsilon) Polyclonal Antibody, Unconjugated (orb13223) at 1:500 overnight at 4C, followed by a conjugated secondary for 20 minutes and 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 (14-3-3 Alpha + Beta + Gamma + Delta + Epsilon) Polyclonal Antibody, Unconjugated 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 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 (14-3-3 Alpha + Beta + Gamma + Delta + Epsilon) Polyclonal Antibody, Unconjugated at 1:200 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining. |
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Sample: Brain Cell Lysate at 40 ug, Primary: Anti-14-3-3 Alpha + Beta + Gamma + Delta + Epsilon (orb13223) at 1/300 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 27 kD, Observed band size: 27 kD. |
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Sample: Cerebrum (Mouse) Lysate at 40 ug, Primary: Anti-14-3-3 Alpha + Beta + Gamma + Delta + Epsilon (orb13223) at 1/300 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 27 kD, Observed band size: 27 kD. |
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Sample: Lane 1: Cerebrum (Mouse) Lysate at 40 ug, Lane 2: Uterus (Mouse) Lysate at 40 ug, Lane 3: Testis (Mouse) Lysate at 40 ug, Lane 4: Liver (Mouse) Lysate at 40 ug, Lane 5: Lung (Mouse) Lysate at 40 ug, Lane 6: Cerebrum (Rat) Lysate at 40 ug, Lane 7: Uterus (Rat) Lysate at 40 ug, Lane 8: Testis (Rat) Lysate at 40 ug, Lane 9: Liver (Rat) Lysate at 40 ug, Lane 10: Lung (Rat) Lysate at 40 ug, Lane 11: NIH/3T3 (Mouse) Cell Lysate at 30 ug, Lane 12: Siha (Human) Cell Lysate at 30 ug, Lane 13: U251 (Human) Cell Lysate at 30 ug, Primary: Anti-14-3-3 Alpha+Beta+Gamma+Delta+Epsilon (orb13 |