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RabMAb

Anti-S6K1 抗体 [E343] (ab32529)

製品の概要

  • 製品名
    Anti-S6K1 antibody [E343]
    S6K1 一次抗体 製品一覧
  • 製品の詳細
    Rabbit monoclonal [E343] to S6K1
  • 由来種
    Rabbit
  • 特異性
    This antibody detects both alpha I and alpha II isoforms.
  • アプリケーション
    適用あり: ICC/IF, WB, IHC-P, Flow Cyt, IPmore details
  • 種交差性
    交差種: Mouse, Rat, Human
  • 免疫原

    Synthetic peptide within Human S6K1 aa 1-100 (N terminal). The exact sequence is proprietary.

  • ポジティブ・コントロール
    • WB: WT HAPI, MCF7 and HEK293 cell lysates. ICC/IF: HeLa cells. Flow Cyt: HeLa, 293T, Neuro-2a and C6 cells. IHC-P Human breast cancer, mouse testis and rat brain tissue.
  • 特記事項

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

    This product is a recombinant rabbit monoclonal antibody.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab32529 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
ICC/IF 1/500.
WB 1/5000 - 1/10000. Detects a band of approximately 70 kDa (predicted molecular weight: 59 kDa). For Rat and Mouse samples 1/500 dilution has only been tried. We have not tested if similarly to Human samples a lot higher dilutions can be used.
IHC-P 1/500.
Flow Cyt 1/100 - 1/2200.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

IP 1/80.

ターゲット情報

  • 機能
    Acts to integrate nutrient and growth factor signals in regulation of protein synthesis, cell proliferation, cell growth, cell cycle progression and cell survival. Downstream effector of the mTOR signaling pathway. Phosphorylates specifically ribosomal protein S6 in response to insulin or several classes of mitogens. During translation initiation, the inactive form associatess with the eIF-3 complex under conditions of nutrient depletion. Mitogenic stimulation leads to phosphorylation and dissociation from the eIF-3 complex and the free activated form can phosphorylate other translational targets including EIF4B. Promotes protein synthesis by phosphorylating PDCD4 at 'Ser-67' and targeting it for degradation. Phosphorylates RICTOR leading to regulation of mammalian target of rapamycin complex 2 (mTORC2) signaling; probably phosphorylates RICTOR at 'Thr-1135'. Phosphorylates IRS1 at multiple serine residues coupled with insulin resistance; probably phosphorylates IRS1 at 'Ser-270'. Required for TNF-alpha induced IRS-1 degradation. Phosphorylates EEF2K in response to IGF1 and inhibits EEF2K activity. Phosphorylates BAD at 'Ser-99' in response to IGF1 leading to BAD inactivation and inhibition of BAD-induced apoptosis. Phosphorylates mitochondrial RMP leading to dissociation of a RMP:PPP1CC complex; probably phosphorylates RMP at 'Ser-99'. The free mitochondrial PPP1CC can dephosphorylate RPS6KB1 at Thr-412 which is proposed to be a negative feed back mechanism for the RPS6KB1 antiapoptotic function. Phosphorylates GSK3B at 'Ser-9' under conditions leading to loss of the TSC1-TSC2 complex. Phosphorylates POLDIP3.
  • 組織特異性
    Widely expressed.
  • 配列類似性
    Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. S6 kinase subfamily.
    Contains 1 AGC-kinase C-terminal domain.
    Contains 1 protein kinase domain.
  • ドメイン
    The autoinhibitory domain is believed to block phosphorylation within the AGC-kinase C-terminal domain and the activation loop.
    The TOS (TOR signaling) motif is essential for activation by mTORC1.
  • 翻訳後修飾
    Phosphorylation at Thr-412 is regulated by mTORC1. The phosphorylation at this site is maintained by an agonist-dependent autophosphorylation mechanism.
  • 細胞内局在
    Cytoplasm; Nucleus. Cytoplasm and Cell junction > synapse > synaptosome. Mitochondrion outer membrane.
  • Information by UniProt
  • 参照データベース
  • 別名
    • 70 kDa ribosomal protein S6 kinase 1 antibody
    • KS6B1_HUMAN antibody
    • p70 alpha antibody
    • P70 beta 1 antibody
    • p70 ribosomal S6 kinase alpha antibody
    • p70 ribosomal S6 kinase beta 1 antibody
    • p70 S6 kinase alpha antibody
    • P70 S6 Kinase antibody
    • p70 S6 kinase, alpha 1 antibody
    • p70 S6 kinase, alpha 2 antibody
    • p70 S6K antibody
    • p70 S6K-alpha antibody
    • p70 S6KA antibody
    • p70(S6K) alpha antibody
    • p70(S6K)-alpha antibody
    • p70-alpha antibody
    • p70-S6K 1 antibody
    • p70-S6K antibody
    • P70S6K antibody
    • P70S6K1 antibody
    • p70S6Kb antibody
    • PS6K antibody
    • Ribosomal protein S6 kinase 70kDa polypeptide 1 antibody
    • Ribosomal protein S6 kinase beta 1 antibody
    • Ribosomal protein S6 kinase beta-1 antibody
    • Ribosomal protein S6 kinase I antibody
    • RPS6KB1 antibody
    • S6K antibody
    • S6K-beta-1 antibody
    • S6K1 antibody
    • Serine/threonine kinase 14 alpha antibody
    • Serine/threonine-protein kinase 14A antibody
    • STK14A antibody
    see all

画像

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: S6K1 knockout HAP1 cell lysate (20 µg)
    Lane 3: MCF7 cell lysate (20 µg)
    Lane 4: HEK293 cell lysate (20 µg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab32529 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.
    ab32529 was shown to recognize S6K1 when S6K1 knockout samples were used, along with additional cross-reactive bands. Wild-type and S6K1 knockout samples were subjected to SDS-PAGE. ab32529 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

  • All lanes : Anti-S6K1 antibody [E343] (ab32529) at 0.004 µg/ml (purified)

    Lane 1 : Neuro2a (Mouse neuroblastoma neuroblast) whole cell lysate
    Lane 2 : Mouse cerebellum lysate
    Lane 3 : C6 (Rat glial tumor glial cell) whole cell lysate
    Lane 4 : Rat cerebellum

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 59 kDa
    Observed band size: 70 kDa (why is the actual band size different from the predicted?)


    Exposure time: 3 minutes


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

  • Immunohistochemical analysis of Human breast cancer tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

  • Immunocytochemistry/Immunofluorescence analysis of C6 cells (Rat glial tumor glial cell)  labelling S6K1 with ab32529 at a dilution of 1:200, 11.1 µg/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A 1:1000 dilution (2μg/ml) was used for the secondary antibody Goat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained with 1:200, 2.5μg/ml with Ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.

  • Immunohistochemical analysis of mouse testis tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

  • Immunohistochemical analysis of rat brain tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

  • Flow cytometry analysis of Neuro-2a (Mouse neuroblastoma neuroblast) cells labelling with ab32529 (purified) at 1/2200 dilution (1 µg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

  • Flow cytometry analysis of 293T (Human embryonic kidney epithelial cell) cells labelling with ab32529 (purified) at 1/2200 dilution (1 µg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

  • Flow cytometry analysis of C6 (Rat glial tumor glial cell) cells labelling with ab32529 (purified) at 1/2200 dilution (1 µg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

  • Lane 1: Neuro2a (Mouse neuroblastoma neuroblast) whole cell lysate, 10µg
    Lane 2:
    Neuro2a whole cell lysate 350µg and ab32529, 2µg
    Lane 3:
    Neuro2a cell lysate, 350µg and rabbit IgG (ab172730), 2µg

    Purified ab32529 immunoprecipitating S6K1 in HEK293T cell lysates. Primary antibody was used at a 1:500 dilution (4.4 μg/ml). For western blotting, VeriBlot for IP (HRP) ab131366 was used as the secondary antibody at 1:1000 dilution.

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

  • Lane 1: HEK293T (Human embryonic kidney epithelial cell) whole cell lysate, 10µg
    Lane 2:
    HEK293T whole cell lysate, 10µg and ab32529, 2µg
    Lane 3:
    HEK293T cell lysate, 350µg and rabbit IgG (ab172730) , 2µg

    Purified ab32529 immunoprecipitating S6K1 in HEK293T cell lysates. Primary antibody was used at a 1:500 dilution (4.4 μg/ml). For western blotting, VeriBlot for IP (HRP) ab131366 was used as the secondary antibody at 1:1000 dilution.

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

  • Immunocytochemistry/Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) labelling with ab32529 at a dilution of 1:200, 11.1 μg/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A 1:1000 dilution (2μg/ml) was used for the secondary antibody Goat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained at 1:200 dilution, 2.5μg/ml with Ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.

  • Immunocytochemistry/Immunofluorescence analysis of MCF 7 (Human breast adenocarcinoma epithelial cell) labeling S6K1 with ab32529 at a dilution of 1:200, 11.1 ug/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A dilution of 1/1000 (2μg/ml)  was used for the secondary antibodyGoat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained at 1:200 dilution, 2.5μg/ml with Ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) . Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling S6K1 with ab32529 at a dilution of 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. ab150077 at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI (blue). 
     
    Confocal image showing cytoplamic staining on HeLa cell line.
  • Overlay histogram showing HeLa cells stained with ab32529 (red line). The cells were fixed with 80% methanol (5 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 (ab32529, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
  • Anti-S6K1 antibody [E343] (ab32529) at 1/10000 dilution + 293T cell lysate

    Predicted band size: 59 kDa
    Observed band size: 70 kDa (why is the actual band size different from the predicted?)

参考文献

This product has been referenced in:

See all 22 Publications for this product

レビューと Q&A

Dear Maren, Thank you for your enquiry. The immunogen sequence information for ab32529 (p70S6 kinase) is proprietary. However, I can provide you with the following information: The immunogen is a 17 aa long peptide that lies in the N-termina...

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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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