Phospho-Akt1 (Ser473) Recombinant Rabbit Monoclonal Antibody

• Catalog Number: orb526646
• Category: Antibodies
• Description: Phospho-Akt1 (Ser473) Recombinant Rabbit Monoclonal Antibody
• Species/Host: Rabbit
• Clonality: Recombinant
• Clone Number: 12A1
• Tested applications: FC, ICC, IF, IHC-Fr, IHC-P, WB
• Predicted Reactivity: Mouse
• Reactivity: Human, Mouse, Rat
• Isotype: IgG
• Immunogen: KLH conjugated Synthesised phosphopeptide derived from human Akt1 around the phosphorylation site of Ser473 QF(p-S)YS
• Concentration: 1mg/ml
• Dilution range: WB=1:500-2000, IHC-P=1:50-200, IHC-F=1:50-200, ICC/IF=1:50-200, IF=1:50-200, Flow-Cyt=2ug/Test
• Form/Appearance: Liquid
• Conjugation: Unconjugated
• MW: 56 kDa
• Target: AKT1
• UniProt ID: P31749
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Buffer/Preservatives: 0.01M TBS (pH7.4) with 1% rAlbumin, 0.02% Proclin300 and 50% Glycerol.
• Alternative names: Akt(Phospho-Ser473); AKT (phospho-S473); AKT (phos
• Note: For research use only
• Expiration Date: 12 months from date of receipt.

Blank control: MCF7. Primary Antibody (green line): Rabbit Anti-Phospho-Akt1 (Ser473) antibody (orb526646), dilution: 2 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 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. Acquisition of 20000 events was performed.

Blank control: U-937. Primary Antibody (green line): Rabbit Anti-Phospho-Akt1 (Ser473) antibody (orb526646), dilution: 2 ug/Test, Secondary Antibody: Goat anti-rabbit IgG-FITC, dilution: 0.5 ug/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. Acquisition of 20000 events was performed.

NIH/3T3 cell, 4% Paraformaldehyde-fixed, Triton X-100 at room temperature for 20 min, Blocking buffer (normal goat serum) at 37°C for 20 min, Antibody incubation with (Phospho-Akt1 (Ser473)) monoclonal Antibody, Unconjugated (orb526646) 1:50, 90 minutes at 37°C, followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nuclei.

Paraformaldehyde-fixed, paraffin embedded (mouse brain), Antigen retrieval by microwaving in EDTA buffer (Ph8.0) for 5 min, Block endogenous peroxidase by 3% hydrogen peroxide for 20 minutes, Blocking buffer (normal goat serum) at 37°C for 30 min, Antibody incubation with (Phospho-Akt1 (Ser473)) Monoclonal Antibody, Unconjugated (orb526646) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse lung), 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 37°C for 30 min, Antibody incubation with (Phospho-Akt1 (Ser473)) Monoclonal Antibody, Unconjugated (orb526646) at 1:50 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Paraformaldehyde-fixed, paraffin embedded (mouse testis), 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 37°C for 30 min, Antibody incubation with (Phospho-Akt1 (Ser473)) Polyclonal Antibody, Unconjugated (orb526646) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Sample: Lane 1: Human HeLa cell lysates, Lane 2: Human Hela treated with 100nM Calyculin A for 30 min, Lane 3: Human MCF-7 cell lysates, Lane 4: Human MCF-7 treated with 100nM Calyculin A for 30 min, Primary: Anti-Phospho-Akt1 (Ser473) (orb526646) at 1/1000 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 56 kDa, Observed band size: 62 kDa.

Sample: Lane 1: Human Jurkat cell lysates, Lane 2: Human MOLT4 cell lysates, Primary: Anti-Phospho-Akt1 (Ser473) (orb526646) at 1/300 dilution, Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution, Predicted band size: 56 kDa, Observed band size: 60 kDa.