Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-S6K1 antibody [E343] - BSA and Azide free (ab203558)

Overview

  • Product name
    Anti-S6K1 antibody [E343] - BSA and Azide free
    See all S6K1 primary antibodies
  • Description
    Rabbit monoclonal [E343] to S6K1 - BSA and Azide free
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IP, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

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

  • General notes

    The formulation and the concentration of this product is compatible for metal-conjugation for mass cytometry (CyTOF®).

    ab203558 is a PBS-only buffer format of ab32529. Please refer to ab32529 for recommended dilutions, protocols, and image data.

    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.

    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.

Properties

Applications

Our Abpromise guarantee covers the use of ab203558 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
WB Use at an assay dependent concentration. 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.

IP Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF Use at an assay dependent concentration.

Target

  • Function
    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.
  • Tissue specificity
    Widely expressed.
  • Sequence similarities
    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.
  • Domain
    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.
  • Post-translational
    modifications
    Phosphorylation at Thr-412 is regulated by mTORC1. The phosphorylation at this site is maintained by an agonist-dependent autophosphorylation mechanism.
  • Cellular localization
    Cytoplasm; Nucleus. Cytoplasm and Cell junction > synapse > synaptosome. Mitochondrion outer membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • 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

Images

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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 Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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 Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

  • 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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

References

ab203558 has not yet been referenced specifically in any publications.

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