Phospho-PKC alpha (T497) Recombinant Rabbit Monoclonal Antibody [JE44-81]
cat.: HA722278
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, FC
Clonality: Monoclonal
Clone number: JE44-81
Form: Liquid
Storage condition: Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 77 kDa
Isotype: IgG
Immunogen: Synthetic phosphopeptide corresponding to residues surrounding Thr497 of Human PKC alpha.
Positive control: HeLa cell lysate, NIH/3T3 cell lysate, C6 cell lysate, MCF7 cells treated with 100nM PMA for 1 hour, HeLa, NIH/3T3, C6, K-562.
Subcellular location: Cytoplasm, Cell membrane, Mitochondrion membrane, Nucleus.
Recommended Dilutions:
  WB
  IF-Cell
  FC
1:2,000
1:100-1:3,000
1:1,000
Uniprot #: SwissProt: P17252 Human | P20444 Mouse | P05696 Rat
Alternative names: AAG6 Aging associated gene 6 aPKC KPCA_HUMAN PKC alpha PKC-A PKC-alpha PKCA PRKACA PRKCA Protein Kinase C alpha Protein kinase C alpha type
Images
HA722278_1.jpg Fig1: Western blot analysis of Phospho-PKC alpha (T497) on different lysates with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: NIH/3T3 cell lysate
Lane 3: C6 cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 77 kDa
Observed band size: 77 kDa

Exposure time: 1 minute; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722278) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HA722278_2.jpg Fig2: Immunocytochemistry analysis of MCF7 cells treated with 100nM PMA for 1 hour labeling Phospho-PKC alpha (T497) with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/3,000 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/3,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HA722278_3.jpg Fig3: Immunocytochemistry analysis of HeLa cells treated with or without λpp labeling Phospho-PKC alpha (T497) with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HA722278_4.jpg Fig4: Immunocytochemistry analysis of NIH/3T3 cells treated with or without λpp labeling Phospho-PKC alpha (T497) with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HA722278_5.jpg Fig5: Immunocytochemistry analysis of C6 cells treated with or without λpp labeling Phospho-PKC alpha (T497) with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-PKC alpha (T497) antibody (HA722278) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HA722278_6.jpg Fig6: Flow cytometric analysis of K-562 cells labeling Phospho-PKC alpha (T497).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722278, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
HA722278_7.jpg Fig7: Flow cytometric analysis of NIH/3T3 cells labeling Phospho-PKC alpha (T497).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722278, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
HA722278_8.jpg Fig8: Flow cytometric analysis of C6 cells labeling Phospho-PKC alpha (T497).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722278, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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