Anti-PKC alpha 抗体 [Y124]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-PKC alpha antibody [Y124] (AB32376)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PKC alpha antibody [Y124] (AB32376)

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PKC alpha antibody [Y124] (AB32376)

Intracellular 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 Fluorr^® 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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PKC alpha antibody [Y124] (AB32376)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC alpha antibody [Y124] (AB32376)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PKC alpha antibody [Y124] (AB32376)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC alpha antibody [Y124] (AB32376)

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.

• IP

Unknown

Immunoprecipitation - Anti-PKC alpha antibody [Y124] (AB32376)

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)

All lanes:

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

Predicted band size: 76 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-PKC alpha antibody [Y124] (AB32376)

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)

All lanes:

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

Predicted band size: 76 kDa

false

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC alpha antibody [Y124] (AB32376)

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

Image from Hao F et al., J Biol Chem. 2011 May 20;286(20):18104-17. Epub 2011 Mar 18. Fig 1.; doi: 10.1074/jbc.M110.208488; May 20, 2011 The Journal of Biological Chemistry, 286, 18104-18117.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC alpha antibody [Y124] (AB32376)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC alpha antibody [Y124] (AB32376)

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.

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-PKC alpha antibody [Y124] (ab32376)

Predicted band size: 76 kDa

false

• WB

Lab

Western blot - Anti-PKC alpha antibody [Y124] (AB32376)

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

All lanes:

Western blot - Anti-PKC alpha antibody [Y124] (ab32376) at 1/10000 dilution

Lane 1:

K562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 76 kDa

Observed band size: 80 kDa

false

• WB

Lab

Western blot - Anti-PKC alpha antibody [Y124] (AB32376)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PKC alpha antibody [Y124] (ab32376) at 1/5000 dilution

Lane 1:

293 (Human embryonic kidney epithelial cell) whole cell lysates at 15 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 76 kDa

false

• WB

Lab

Western blot - 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 - 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.

All lanes:

Western blot - Anti-PKC alpha antibody [Y124] (ab32376)

Predicted band size: 76 kDa

false

• WB

Lab

Western blot - Anti-PKC alpha antibody [Y124] (AB32376)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-PKC alpha antibody [Y124] (ab32376) at 1/5000 dilution

Lane 1:

C6 (Rat glial tumor glial cell) whole cell lysates at 15 µg

Lane 2:

Pig skeletal muscle lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 76 kDa

false

• WB

Supplier Data

Western blot - Anti-PKC alpha antibody [Y124] (AB32376)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PKC alpha antibody [Y124] (ab32376) at 1/2000 dilution

Lane 1:

Western blot - Recombinant human PKC alpha protein (Active) (<a href='/products/proteins-peptides/recombinant-human-pkc-alpha-protein-active-ab55672'>ab55672</a>)

Lane 2:

Western blot - Recombinant human PKC beta 1 protein (<a href='/products/proteins-peptides/recombinant-human-pkc-beta-1-protein-ab60840'>ab60840</a>)

Lane 3:

Western blot - Recombinant human PKC beta 2 protein (<a href='/products/proteins-peptides/recombinant-human-pkc-beta-2-protein-ab60841'>ab60841</a>)

Lane 4:

Western blot - Recombinant human PKC gamma protein (<a href='/products/proteins-peptides/recombinant-human-pkc-gamma-protein-ab60842'>ab60842</a>)

Lane 5:

Western blot - Recombinant human PKC delta protein (<a href='/products/proteins-peptides/recombinant-human-pkc-delta-protein-ab60844'>ab60844</a>)

Lane 6:

Western blot - Recombinant human PKC epsilon protein (<a href='/products/proteins-peptides/recombinant-human-pkc-epsilon-protein-ab60847'>ab60847</a>)

Lane 7:

Western blot - Recombinant human PKC zeta protein (<a href='/products/proteins-peptides/recombinant-human-pkc-zeta-protein-ab60848'>ab60848</a>)

Lane 8:

Western blot - Recombinant human PKC eta protein (<a href='/products/proteins-peptides/recombinant-human-pkc-eta-protein-ab60849'>ab60849</a>)

Lane 9:

Western blot - Recombinant human PKC theta/PRKCQ protein (<a href='/products/proteins-peptides/recombinant-human-pkc-theta-prkcq-protein-ab56641'>ab56641</a>)

Lane 10:

Western blot - Recombinant human PKC iota protein (<a href='/products/proteins-peptides/recombinant-human-pkc-iota-protein-ab60850'>ab60850</a>)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 76 kDa

Observed band size: 105 kDa

false

Exposure time: 20s