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RabMAb

Anti-PKC alpha抗体[Y124] (ab32376)

概述

  • 产品名称
    Anti-PKC alpha抗体[Y124]
    参阅全部 PKC alpha 一抗
  • 描述
    兔单克隆抗体[Y124] to PKC alpha
  • 经测试应用
    适用于: WB, IHC-P, Flow Cyt, IP, ICC/IF, IHC-Frmore details
  • 种属反应性
    与反应: Mouse, Rat, Human, Pig, Goldfish
  • 免疫原

    within Human PKC alpha aa 650 to the C-terminus (C terminal). The exact sequence is proprietary.

  • 阳性对照
    • 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.
  • 常规说明

    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.

性能

应用

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.

应用 Ab评论 说明
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. 

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.

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.

IHC-Fr 1/100.

靶标

  • 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
  • 数据库链接
  • 别名
    • AAG6 antibody
    • Aging associated gene 6 antibody
    • aPKC antibody
    • KPCA_HUMAN antibody
    • PKC alpha antibody
    • PKC-A antibody
    • PKC-alpha antibody
    • PKCA antibody
    • PRKACA antibody
    • PRKCA antibody
    • Protein Kinase C alpha antibody
    • Protein kinase C alpha type antibody
    see all

图片



  • Predicted band size : 77 kDa

    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.

  • 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)

  • 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).



  • Predicted band size : 77 kDa

    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 unpurified 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.

  • 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
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size : 77 kDa

    Blocking and diluting buffer: 5% NFDM/TBST

  • 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
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Predicted band size : 77 kDa

    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.

  • 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
    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)

  • Unpurified ab32376, at a 1/250 dilution, staining PKC alpha in HeLa cells by Immunofluorescence.

  • Anti-PKC alpha antibody [Y124] (ab32376) at 1/10000 dilution (unpurified) + 293 cell lysate

    Predicted band size : 77 kDa
    Observed band size : 75 kDa (why is the actual band size different from the predicted?)

文献

This product has been referenced in:
  • Ono A  et al. Feedback activation of AMPK-mediated autophagy acceleration is a key resistance mechanism against SCD1 inhibitor-induced cell growth inhibition. PLoS One 12:e0181243 (2017). Read more (PubMed: 28704514) »
  • Zhang HM  et al. Moderate lifelong overexpression of tuberous sclerosis complex 1 (TSC1) improves health and survival in mice. Sci Rep 7:834 (2017). WB . Read more (PubMed: 28400571) »

See all 29 Publications for this product

客户评价及客户问答

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Human umbilical vein endothelial cell)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (10%)
Loading amount
20 µg
Specification
Human umbilical vein endothelial cell
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

提交于 Nov 07 2017

Application
Immunohistochemistry (Frozen sections)
Sample
Zebrafish Cell (5 um frozen retinal section)
Permeabilization
No
Specification
5 um frozen retinal section
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 20% · Temperature: 25°C
Fixative
Paraformaldehyde
Username

Dr. Jenny Lenkowski

Verified customer

提交于 Aug 02 2017

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Fukomys anselli Tissue sections (Retina)
Antigen retrieval step
None
Permeabilization
No
Specification
Retina
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 21°C
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

提交于 Dec 21 2016

Application
Western blot
Sample
Rat Tissue lysate - whole (heart)
Gel Running Conditions
Reduced Denaturing (9%)
Loading amount
60 µg
Treatment
TNF-a for 30in
Specification
heart
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Mr. Baptiste Jude

Verified customer

提交于 Jul 07 2016

Sorry for the delay in getting back to you.

The peptide used to raise the anti-PKC alpha antibody [Y124] (ab32376) has now been added to the catalogue for you. This is under the catalogue number ab133953.

I hope this has been of hel...

Read More

Thank you for your reply.

I will organise the peptide to be added to the catalogue for you. Once this has been done I will let you know.

If youhave any furtherquestions please do not hesitate to contact us.

Thank you for contacting us on Friday in regards to the immunogen used to raise the anti-PKC alpha antibody [Y124] (ab32376).

I am please do say that I am able to share some information regarding the immunogen sequence. I cannot give you the ...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (MCF7)
Loading amount
100000 cells
Specification
MCF7
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C
Username

Abcam user community

Verified customer

提交于 May 17 2012

According to our records, ab32336 was proving difficult to use inWB and we were in contact in order to help resolve the issue. Looking at our correspondence, it appears that we are awaiting more details in order to help us better understand the difficu...

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Thank you for contacting us. I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. I am atta...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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