Recombinant Anti-PKC alpha antibody [Y124] (ab32376)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PKC alpha knockout HAP1 cell lysate (20 µg)
Lane 3: K562 cell lysate (20 µg)
Lane 4: HEK293 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab32376 observed at 77 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32376 was shown to specifically react with PKC alpha in wild-type HAP1 cells. No band was observed when PKC alpha knockout samples were examined. Wild-type and PKC alpha knockout samples were subjected to SDS-PAGE. ab32376 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/1000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Unpurified ab32376 staining PKCα in 200nM PMA-treated wild-type HAP1 cells (top panel) and PKCα in 200nM PMA-treated knockout HAP1 cells (bottom panel). The cells were treated with 200nM PMA for 30 minutes to induce translocation of PKCα to the cell membrane. The cells were then fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32376 at 1/200 dilution and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150117) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal under the same testing conditions in HAP1 cells fixed with 4% formaldehyde (10 min).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab32376 (purified) at 1:20 dilution (0.5ug) immunoprecipitating PKC alpha in 293 (Human embryonic kidney epithelial cell) whole cell lysate.

Lane 1 (input): 293 (Human embryonic kidney epithelial cell) whole cell lysate 10ug

Lane 2 (+): ab32376 & 293 (Human embryonic kidney epithelial cell) whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32376 in 293 (Human embryonic kidney epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

The band between 37kDa and 50kDa might be the C-term fragment. (PMID:10381525)

ab32376 (purified) at 1:20 dilution (0.5ug) immunoprecipitating PKC alpha in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate.

Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10ug

Lane 2 (+): ab32376 & Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32376 in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

The band between 37kDa and 50kDa might be the C-term fragment. (PMID:10381525)

Unpurified ab32376 staining PKCα in wild-type HAP1 cells (top panel) and PKCα in knockout HAP1 cells (bottom panel). In untreated conditions, PKCα is expressed in the cytoplasm of the cells. The cells were then fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32376 at 1/200 dilution and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150117) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal under the same testing conditions in HAP1 cells fixed with 4% formaldehyde (10 min).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: PKC alpha knockout HAP1 cell lysate (20 µg)
Lane 3: K562 cell lysate (20 µg)
Lane 4: HEK293 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - unpurified ab32376 observed at 77 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab32376 and a competitor's top cited rabbit polyclonal antibody.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PKC alpha with purified ab32376 at 1:20 dilution (5 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1:2000 dilution. Isotype control - 90% methanol. Unlabeled control - Rabbit monoclonal IgG (Black). Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PKC alpha with Purified ab32376 at 1:250 dilution (0.4μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat liver tissue sections labeling PKC alpha with purified  ab32376 at 1:100 dilution (1.01 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human glioma tissue sections labeling PKC alpha with purified  ab32376 at 1:100 dilution (1.01 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse liver tissue sections labeling PKC alpha with purified  ab32376 at 1:100 dilution (1.01 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human endometrium carcinoma tissue sections labeling PKC alpha with purified ab32376 at 1:100 dilution (1.01 μg/ml). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using EDTA Buffer, pH9.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Blocking and diluting buffer: 5% NFDM/TBST

Blocking and diluting buffer: 5% NFDM/TBST

Overlay histogram showing HeLa cells stained with unpurified ab32376 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32376, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

Immunohistochemical analysis of mouse small intestine tissue, staining PKC alpha with unpurified ab32376.

Blocking and diluting buffer: 5% NFDM/TBST 
The band between 37kDa and 50kDa might be the C-term fragment. (PMID:10381525)

ab32376 staining PKC in the HT1080 Cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody (1/400 in PBS) for 1 hour at 22°C. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody (1/1000).

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