Anti-PKC alpha 抗体 [Y124] (ab32376)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y124] to PKC alpha
- Suitable for: Flow Cyt (Intra), WB, IHC-P, IP, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human, Pig
Related conjugates and formulations
製品の概要
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製品名
Anti-PKC alpha antibody [Y124]
PKC alpha 一次抗体 製品一覧 -
製品の詳細
Rabbit monoclonal [Y124] to PKC alpha -
由来種
Rabbit -
アプリケーション
適用あり: Flow Cyt (Intra), WB, IHC-P, IP, ICC/IFmore details -
種交差性
交差種: Mouse, Rat, Human, Pig
交差が予測される動物種: Goldfish -
免疫原
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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ポジティブ・コントロール
- 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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特記事項
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
製品の特性
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製品の状態
Liquid -
保存方法
Shipped 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. -
バッファー
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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精製度
Protein A purified -
ポリ/モノ
モノクローナル -
クローン名
Y124 -
アイソタイプ
IgG -
研究分野
関連製品
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Alternative Versions
- Alexa Fluor® 488 Anti-PKC alpha antibody [Y124] (ab204633)
- Alexa Fluor® 647 Anti-PKC alpha antibody [Y124] (ab204634)
- HRP Anti-PKC alpha antibody [Y124] (ab205422)
- Alexa Fluor® 594 Anti-PKC alpha antibody [Y124] (ab206344)
- Alexa Fluor® 405 Anti-PKC alpha antibody [Y124] (ab206345)
- PE Anti-PKC alpha antibody [Y124] (ab208749)
- 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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Recombinant Protein
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Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab32376の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
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Flow Cyt (Intra) |
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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WB | (6) |
1/1000 - 1/10000. Detects a band of approximately 75 kDa (predicted molecular weight: 77 kDa).
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IHC-P |
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
For unpurified use at 1/250 - 1/500. |
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IP |
1/20.
For unpurified use at 1/100. |
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ICC/IF | (2) |
1/200.
Treatment with 200nM PMA for 30 minutes induces translocation of PKC alpha to the membrane. |
特記事項 |
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Flow Cyt (Intra)
1/20. For unpurified use at 1/100. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
1/1000 - 1/10000. Detects a band of approximately 75 kDa (predicted molecular weight: 77 kDa). |
IHC-P
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. For unpurified use at 1/250 - 1/500. |
IP
1/20. For unpurified use at 1/100. |
ICC/IF
1/200. Treatment with 200nM PMA for 30 minutes induces translocation of PKC alpha to the membrane. |
ターゲット情報
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機能
This 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. -
配列類似性
Belongs 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. -
細胞内局在
Cytoplasm. Cell membrane. Nucleus. - Information by UniProt
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参照データベース
- 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 -
別名
- AAG6 antibody
- Aging associated gene 6 antibody
- aPKC antibody
see all
画像
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All lanes : Anti-PKC alpha antibody [Y124] (ab32376) at 1/2000 dilution
Lane 1 :Recombinant human PKC alpha protein (Active) (ab55672)
Lane 2 :Recombinant human PKC beta 1 protein (ab60840)
Lane 3 :Recombinant human PKC beta 2 protein (ab60841)
Lane 4 :Recombinant human PKC gamma protein (ab60842)
Lane 5 :Recombinant human PKC delta protein (ab60844)
Lane 6 :Recombinant human PKC epsilon protein (ab60847)
Lane 7 :Recombinant human PKC zeta protein (ab60848)
Lane 8 :Recombinant human PKC eta protein (ab60849)
Lane 9 :Recombinant human PKC theta/PRKCQ protein (ab56641)
Lane 10 :Recombinant human PKC iota protein (ab60850)
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 77 kDa
Observed band size: 105 kDa why is the actual band size different from the predicted?
Exposure time: 20 secondsBlocking and diluting buffer and concentration: 5% NFDM/TBST.
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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 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)
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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 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.
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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).
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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) 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) 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) 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) 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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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) -
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).
プロトコール
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (106)
ab32376 は 106 報の論文で使用されています。
- Wang X et al. SOX2-positive retinal stem cells are identified in adult human pars plicata by single-cell transcriptomic analyses. MedComm (2020) 4:e198 (2023). PubMed: 36582303
- Chen Y et al. Inhibition of protein kinase C alpha attenuates lipopolysaccharide-triggered acute lung injury by alleviating the hyperinflammatory response and oxidative stress. Ann Transl Med 10:132 (2022). PubMed: 35284551
- Hu J et al. A reduction of Syndecan-4 in macrophages promotes atherosclerosis by aggravating the proinflammatory capacity of macrophages. J Transl Med 20:319 (2022). PubMed: 35842658
- van Senten JR et al. Use of CRISPR/Cas9-edited HEK293 cells reveals that both conventional and novel protein kinase C isozymes are involved in mGlu5a receptor internalization. J Biol Chem 298:102466 (2022). PubMed: 36087841
- Cao H et al. PPP1R14D promotes the proliferation, migration and invasion of lung adenocarcinoma via the PKCα/BRAF/MEK/ERK signaling pathway. Int J Oncol 61:N/A (2022). PubMed: 36263632