重组Anti-PKC alpha抗体[Y124] - BSA and Azide free (ab221611)
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 - BSA and Azide free
- Suitable for: Flow Cyt (Intra), WB, IP, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Mouse, Rat, Human, Pig
Related conjugates and formulations
概述
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产品名称
Anti-PKC alpha抗体[Y124] - BSA and Azide free
参阅全部 PKC alpha 一抗 -
描述
兔单克隆抗体[Y124] to PKC alpha - BSA and Azide free -
宿主
Rabbit -
经测试应用
适用于: Flow Cyt (Intra), WB, IP, IHC-P, ICC/IFmore details -
种属反应性
与反应: Mouse, Rat, Human, Pig
预测可用于: Goldfish -
免疫原
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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阳性对照
- WB: 293 cell lysate. IHC-P: Human lung carcinoma tissue. ICC/IF: HeLa and PMA-Treated and untreated wild-type HAP1 cells.
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常规说明
ab221611 is the carrier-free version of ab32376.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
性能
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形式
Liquid -
存放说明
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
存储溶液
pH: 7.20
Constituent: PBS -
无载体
是 -
Concentration information loading...
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纯度
Protein A purified -
克隆
单克隆 -
克隆编号
Y124 -
同种型
IgG -
研究领域
相关产品
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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] (ab32376)
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Compatible Secondaries
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Conjugation kits
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Isotype control
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Positive Controls
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Recombinant Protein
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab221611于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。
应用 | Ab评论 | 说明 |
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Flow Cyt (Intra) |
Use at an assay dependent concentration.
ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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WB |
Use at an assay dependent concentration. Detects a band of approximately 75 kDa (predicted molecular weight: 77 kDa).
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IP |
Use at an assay dependent concentration.
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ICC/IF |
Use at an assay dependent concentration.
Treatment with 200nM PMA for 30 minutes induces translocation of PKC alpha to the membrane. |
说明 |
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Flow Cyt (Intra)
Use at an assay dependent concentration. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
Use at an assay dependent concentration. Detects a band of approximately 75 kDa (predicted molecular weight: 77 kDa). |
IP
Use at an assay dependent concentration. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ICC/IF
Use at an assay dependent concentration. Treatment with 200nM PMA for 30 minutes induces translocation of PKC alpha to the membrane. |
靶标
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功能
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. -
序列相似性
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. -
细胞定位
Cytoplasm. Cell membrane. Nucleus. - Information by UniProt
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数据库链接
- 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 -
别名
- AAG6 antibody
- Aging associated gene 6 antibody
- aPKC antibody
see all
图片
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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 secondsThis data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
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This WB data was generated using the same anti-PKC alpha antibody clone, Y124, in a different buffer formulation (cat# 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.
Unpurified ab32376 was shown to specifically react with PKC alpha when PKC alpha knockout samples were used. 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 h at room temperature before imaging. -
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)This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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)
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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 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).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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).This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32376).
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This ICC/IF data was generated using the same anti-PKC alpha antibody clone, Y124, in a different buffer formulation (cat# 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).
实验方案
数据表及文件
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Datasheet download
Certificate of Compliance
文献 (28)
ab221611 被引用在 28 文献中.
- Jiang Y et al. Aquaporin 1 mediates early responses to osmotic stimuli in endothelial cells via the calmodulin pathway. FEBS Open Bio 11:75-84 (2021). PubMed: 33125833
- Wang XH et al. Cannabinoid CB1 receptor signaling dichotomously modulates inhibitory and excitatory synaptic transmission in rat inner retina. Brain Struct Funct 221:301-16 (2016). PubMed: 25273281
- Lum MA et al. PKCa is Resistant to Long-Term Desensitization/Downregulation by Prolonged Diacylglycerol Stimulation. J Biol Chem N/A:N/A (2016). WB, IHC, ICC/IF ; Rat, Human . PubMed: 26769967
- Termini CM et al. Tetraspanin CD82 Regulates the Spatiotemporal Dynamics of PKCa in Acute Myeloid Leukemia. Sci Rep 6:29859 (2016). IHC-P ; Human . PubMed: 27417454
- Yang GY et al. Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge. Vet Res 47:71 (2016). WB . PubMed: 27424033