Phospho-PKC alpha (Thr638) Recombinant Rabbit Monoclonal Antibody

• Catalog Number: orb559135
• Category: Antibodies
• Description: Phospho-PKC alpha (Thr638) Recombinant Rabbit Monoclonal Antibody
• Species/Host: Rabbit
• Clonality: Recombinant
• Clone Number: 4B3
• Tested applications: FC, ICC, IF, IHC-Fr, IHC-P, WB
• Predicted Reactivity: Bovine, Equine, Gallus, Porcine
• Reactivity: Human, Mouse, Rat
• Isotype: IgG
• Immunogen: KLH conjugated synthesised phosphopeptide derived from human PKC alpha around the phosphorylation site of Thr638 VL(p-T)PP
• Concentration: 1mg/ml
• Dilution range: WB=1:500-2000, IHC-P=1:50-1:500, IHC-F=1:50-1:500, ICC/IF=1:50, IF=1:50-1:500, Flow-Cyt=2ug/Test
• Form/Appearance: Liquid
• Conjugation: Unconjugated
• MW: 77 kDa
• Target: PRKCA
• UniProt ID: P17252
• 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: PKC alpha(T638); PKC(phospho-Thr641); phospho-PKC
• Note: For research use only
• Expiration Date: 12 months from date of receipt.

Blank control: MCF7. Primary Antibody (green line): Rabbit Anti-Phospho-PKC alpha (Thr638) antibody (orb559135), 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.

Hela 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-PKC alpha (T638) in) monoclonal Antibody, Unconjugated (orb559135) 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.

MCF-7 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-PKC alpha (T638) in) monoclonal Antibody, Unconjugated (orb559135) 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 (human breast carcinoma), 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-PKC alpha (Thr638)) Monoclonal Antibody, Unconjugated (orb559135) at 1:50 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

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 37°C for 30 min, Antibody incubation with (Phospho-PKC alpha (Thr638)) Monoclonal Antibody, Unconjugated (orb559135) at 1:50 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

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 37°C for 30 min, Antibody incubation with (Phospho-PKC alpha (Thr638)) Monoclonal Antibody, Unconjugated (orb559135) at 1:50 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.

Sample: Lane 1: 293 cell lysate, Lane 2: Hela cell lysate, Lane 3: Jurkat cell lysate, Primary: Anti-Phospho-PKC alpha (Thr638) (orb559135) at 1/500 dilution, Secondary: Goat Anti-Rabbit IgG - HRP at 1:5000 dilution, Predicted band size: 77 kD, Observed band size: 77 kD.

SH-SY5Y 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-PKC alpha (T638)) monoclonal Antibody, Unconjugated (orb559135) 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.