

Anti-PKC alpha antibody [Y124] (ab32376)
- Datasheet
- References (37)
- Protocols
Overview
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Product nameAnti-PKC alpha antibody [Y124]
See all PKC alpha primary antibodies -
DescriptionRabbit monoclonal [Y124] to PKC alpha
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Host speciesRabbit
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Tested applicationsSuitable for: WB, IHC-P, Flow Cyt, IP, ICC/IF, IHC-Frmore details
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Species reactivityReacts with: Mouse, Rat, Human, Pig, Goldfish
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Immunogen
Synthetic peptide within Human PKC alpha aa 650 to the C-terminus (C terminal). The exact sequence is proprietary.
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Positive control
- WB: HEK293, K562, HeLa, C6 cell lysates and pig skeletal muscle tissue lysates. IHC-P: Human lung carcinoma and human endometrium carcinoma tissues. ICC/IF: PMA-Treated and untreated wild-type HAP1 and HeLa cells.
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General notes
A trial size is available to purchase for this antibody.
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
This product is a recombinant rabbit monoclonal antibody.
Properties
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FormLiquid
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Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
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Storage bufferpH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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PurityProtein A purified
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ClonalityMonoclonal
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Clone numberY124
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IsotypeIgG
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Research areas
Associated products
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Alternative Versions
- Anti-PKC alpha antibody [Y124] (Alexa Fluor® 488) (ab204633)
- Anti-PKC alpha antibody [Y124] (Alexa Fluor® 647) (ab204634)
- Anti-PKC alpha antibody [Y124] (HRP) (ab205422)
- Anti-PKC alpha antibody [Y124] (Alexa Fluor® 594) (ab206344)
- Anti-PKC alpha antibody [Y124] (Alexa Fluor® 405) (ab206345)
- Anti-PKC alpha antibody [Y124] (Phycoerythrin) (ab208749)
- Anti-PKC alpha antibody [Y124] (Alexa Fluor® 568) (ab208827)
- Anti-PKC alpha antibody [Y124] - BSA and Azide free (ab221611)
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Compatible Secondaries
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Isotype control
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Positive Controls
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Related Products
Applications
Our Abpromise guarantee covers the use of ab32376 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB | 1/1000 - 1/10000. Detects a band of approximately 75 kDa (predicted molecular weight: 77 kDa). | |
IHC-P | 1/100. For unpurified use at 1/250 - 1/500. |
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Flow Cyt | 1/20. For unpurified use at 1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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IP | 1/20. For unpurified use at 1/100. |
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ICC/IF | 1/200. Treatment with 200nM PMA for 30 minutes induces translocation of PKC alpha to the membrane. |
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IHC-Fr | 1/100. |
Target
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FunctionThis is a calcium-activated, phospholipid-dependent, serine- and threonine-specific enzyme. May play a role in cell motility by phosphorylating CSPG4.
PKC is activated by diacylglycerol which in turn phosphorylates a range of cellular proteins. PKC also serves as the receptor for phorbol esters, a class of tumor promoters. -
Sequence similaritiesBelongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. PKC subfamily.
Contains 1 AGC-kinase C-terminal domain.
Contains 1 C2 domain.
Contains 2 phorbol-ester/DAG-type zinc fingers.
Contains 1 protein kinase domain. -
Cellular localizationCytoplasm. Cell membrane. Nucleus.
- Information by UniProt
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Database links
- Entrez Gene: 5578 Human
- Entrez Gene: 18750 Mouse
- Entrez Gene: 397184 Pig
- Entrez Gene: 24680 Rat
- Omim: 176960 Human
- SwissProt: P17252 Human
- SwissProt: P20444 Mouse
- SwissProt: P05696 Rat
see all -
Alternative names
- AAG6 antibody
- Aging associated gene 6 antibody
- aPKC antibody
see all
Images
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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.
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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 secondary antibody (HRP) (ab131366) was used as the secondary antibody 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)
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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 secondary antibody (HRP) (ab131366) was used as the secondary antibody 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.
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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).
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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.
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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.
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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.
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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.
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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.
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All lanes : Anti-PKC alpha antibody [Y124] (ab32376) at 1/5000 dilution (purified)
Lane 1 : 293 (Human embryonic kidney epithelial cell) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Predicted band size: 77 kDaBlocking and diluting buffer: 5% NFDM/TBST
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All lanes : Anti-PKC alpha antibody [Y124] (ab32376) at 1/5000 dilution (purified)
Lane 1 : C6 (Rat glial tumor glial cell) whole cell lysates
Lane 2 : Pig skeletal muscle lysates
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Predicted band size: 77 kDaBlocking and diluting buffer: 5% NFDM/TBST
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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.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC alpha antibody [Y124] (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.
Immunohistochemical analysis of mouse small intestine tissue, staining PKC alpha with unpurified ab32376.
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All lanes : Anti-PKC alpha antibody [Y124] (ab32376) at 1/10000 dilution (purified)
Lane 1 : K562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 77 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?Blocking and diluting buffer: 5% NFDM/TBST
The band between 37kDa and 50kDa might be the C-term fragment. (PMID:10381525) -
Immunocytochemistry/ Immunofluorescence - Anti-PKC alpha antibody [Y124] (ab32376)This image is courtesy of an anonymous Abreview
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).
References
This product has been referenced in:
- Ji Y et al. Astragalosides increase the cardiac diastolic function and regulate the "Calcium sensing receptor-protein kinase C-protein phosphatase 1" pathway in rats with heart failure. Biomed Pharmacother 103:838-843 (2018). WB . Read more (PubMed: 29710499) »
- Dandekar MP et al. Intravenous infusion of xenon-containing liposomes generates rapid antidepressant-like effects. Prog Neuropsychopharmacol Biol Psychiatry 86:140-149 (2018). WB . Read more (PubMed: 29559371) »