Blank control (blue line): Hela (fixed with 70% methanol (Overnight at 4C) and then permeabilized with 90% ice-cold methanol for 20 min at -20C). Primary Antibody (green line): Rabbit Anti-phospho-IKB alpha (Tyr42) antibody (orb5520), 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.
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 (Tyr42) antibody (orb5520), 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.
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 (p-IKB alpha (Tyr42)) Polyclonal Antibody, Unconjugated (orb5520) at 1:400 overnight at 4C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
Sample: Lane 1: HepG2 (Human) Cell Lysate at 30 ug, Lane 2: Hela (Human) Cell Lysate at 30 ug, Primary: Anti-phospho-IKB alpha (Tyr42) (orb5520) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 39 kD, Observed band size: 37 kD.
Sample: Raji (Human) Lysate at 40 ug, Primary: Anti-phospho-IKB alpha (Tyr42) (orb5520) at 1/300 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 35 kD, Observed band size: 35 kD.
Sample: Thymus (Mouse) Lysate at 40 ug, Primary: Anti-phospho-IKB alpha (Tyr42) (orb5520) at 1/300 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 35 kD, Observed band size: 35 kD.
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