Anti-AKT1 (phospho S473) 抗体 [EP2109Y] (ab81283)

製品の概要

  • 製品名Anti-AKT1 (phospho S473) antibody [EP2109Y]
    AKT1 一次抗体 製品一覧
  • 製品の詳細
    Rabbit monoclonal [EP2109Y] to AKT1 (phospho S473)
  • 特異性ab81283 detects AKT1 phosphorylated at Serine 473. The region of AKT1 surrounding S473 has a high degree of similarity to the corresponding regions in AKT2 and AKT3 and thus may cross react with these proteins if phosphorylated on the corresponding serine residue.
  • アプリケーション適用あり: ICC/IF, IHC-Fr, IHC-P, WBmore details
    適用なし: Flow Cyt or IP
  • 種交差性
    交差種: Mouse, Rat, Human
  • 免疫原

    A phospho specific peptide corresponding to residues surrounding Ser473 of human AKT1.

  • ポジティブ・コントロール
    • 3T3 cell lysate treated with PDGF. Cervical carcinoma.
  • 特記事項

    This product is a recombinant rabbit monoclonal antibody.

     

    Produced using Abcam’s RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487.

    A trial size is available to purchase for this antibody.

     

    Alternative versions available:

    Anti-AKT1 (phospho S473) antibody (Alexa Fluor® 488) [EP2109Y] (ab194198)
    Anti-AKT1 (phospho S473) antibody (Alexa Fluor® 647) [EP2109Y] (ab194200)
    Anti-AKT1 (phospho S473) antibody (HRP) [EP2109Y] (ab194201)

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab81283 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 1/100 - 1/250.
IHC-Fr Use at an assay dependent concentration.
IHC-P 1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB 1/5000 - 1/10000. Predicted molecular weight: 56 kDa.Can be blocked with AKT1 peptide (ab171724) or AKT1 peptide (ab217601).

Abcam recommends using BSA as the blocking agent.

  • 追加情報Is unsuitable for Flow Cyt or IP.
  • ターゲット情報

    • 機能Plays a role as a key modulator of the AKT-mTOR signaling pathway controlling the tempo of the process of newborn neurons integration during adult neurogenesis, including correct neuron positioning, dendritic development and synapse formation (By similarity). General protein kinase capable of phosphorylating several known proteins. Phosphorylates TBC1D4. Signals downstream of phosphatidylinositol 3-kinase (PI(3)K) to mediate the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor I (IGF-I). Plays a role in glucose transport by mediating insulin-induced translocation of the GLUT4 glucose transporter to the cell surface. Mediates the antiapoptotic effects of IGF-I. Mediates insulin-stimulated protein synthesis by phosphorylating TSC2 at 'Ser-939' and 'Thr-1462', thereby activating mTORC1 signaling and leading to both phosphorylation of 4E-BP1 and in activation of RPS6KB1. Promotes glycogen synthesis by mediating the insulin-induced activation of glycogen synthase. The activated form can suppress FoxO gene transcription and promote cell cycle progression. Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly.
    • 組織特異性Expressed in all human cell types so far analyzed. The Tyr-176 phosphorylated form shows a significant increase in expression in breast cancers during the progressive stages i.e. normal to hyperplasia (ADH), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC) and lymph node metastatic (LNMM) stages.
    • 関連疾患Defects in AKT1 are a cause of susceptibility to breast cancer (BC) [MIM:114480]. A common malignancy originating from breast epithelial tissue. Breast neoplasms can be distinguished by their histologic pattern. Invasive ductal carcinoma is by far the most common type. Breast cancer is etiologically and genetically heterogeneous. Important genetic factors have been indicated by familial occurrence and bilateral involvement. Mutations at more than one locus can be involved in different families or even in the same case.
      Defects in AKT1 are associated with colorectal cancer (CRC) [MIM:114500].
      Defects in AKT1 are associated with susceptibility to ovarian cancer [MIM:604370]; also called susceptibility to familial breast-ovarian cancer type 1 (BROVCA1).
    • 配列類似性Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. RAC subfamily.
      Contains 1 AGC-kinase C-terminal domain.
      Contains 1 PH domain.
      Contains 1 protein kinase domain.
    • ドメインBinding of the PH domain to the phosphatidylinositol 3-kinase alpha (PI(3)K) results in its targeting to the plasma membrane. The PH domain mediates interaction with TNK2 and Tyr-176 is also essential for this interaction.
      The AGC-kinase C-terminal mediates interaction with THEM4.
    • 翻訳後修飾Phosphorylation on Thr-308, Ser-473 and Tyr-474 is required for full activity. Activated TNK2 phosphorylates it on Tyr-176 resulting in its binding to the anionic plasma membrane phospholipid PA. This phosphorylated form localizes to the cell membrane, where it is targeted by PDPK1 and PDPK2 for further phosphorylations on Thr-308 and Ser-473 leading to its activation. Ser-473 phosphorylation by mTORC2 favors Thr-308 phosphorylation by PDPK1. Ser-473 phosphorylation is enhanced by interaction with AGAP2 isoform 2 (PIKE-A). Ser-473 phosphorylation is enhanced in focal cortical dysplasias with Taylor-type balloon cells.
      Ubiquitinated; undergoes both 'Lys-48'- and 'Lys-63'-linked polyubiquitination. TRAF6-induced 'Lys-63'-linked AKT1 ubiquitination is critical for phosphorylation and activation. When ubiquitinated, it translocates to the plasma membrane, where it becomes phosphorylated. When fully phosphorylated and translocated into the nucleus, undergoes 'Lys-48'-polyubiquitination catalyzed by TTC3, leading to its degradation by the proteasome.
    • 細胞内局在Cytoplasm. Nucleus. Cell membrane. Nucleus after activation by integrin-linked protein kinase 1 (ILK1). Nuclear translocation is enhanced by interaction with TCL1A. Phosphorylation on Tyr-176 by TNK2 results in its localization to the cell membrane where it is targeted for further phosphorylations on Thr-308 and Ser-473 leading to its activation and the activated form translocates to the nucleus.
    • Information by UniProt
    • 参照データベース
    • 別名
      • AKT 1 antibody
      • AKT antibody
      • AKT1 antibody
      • AKT1_HUMAN antibody
      • MGC99656 antibody
      • PKB antibody
      • PKB-ALPHA antibody
      • PRKBA antibody
      • Protein Kinase B Alpha antibody
      • Protein kinase B antibody
      • Proto-oncogene c-Akt antibody
      • RAC Alpha antibody
      • RAC antibody
      • RAC-alpha serine/threonine-protein kinase antibody
      • RAC-PK-alpha antibody
      see all

    Anti-AKT1 (phospho S473) antibody [EP2109Y] 画像

    • Immunohistochemical analysis of Human HPV16 immortalized keratinocytes transfected with non-targeting siRNA, staining AKT1 (phospho S473) (green) with ab81283.
      Antigen retrieval was performed by heat mediation in citrate buffer (pH 6). Samples were blocked with 10% goat serum before incubating with primary antibody (1/100). Fluoroscein-conjugated tyramide was used to detect staining.

    • ab81283, at 1/100 dilution, staining AKT1 in untreated (left panel) and Phosphatase-treated (right panel) cervical carcinoma by Immunohistochemistry using formalin-fixed, paraffin-embedded tissue
    • All lanes : Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) at 1/1000 dilution

      Lane 1 : HEK293 cell lysate
      Lane 2 : HEK293 cell lysate
      Lane 3 : HEK293 cell lysate


      Predicted band size : 56 kDa

      Insulin treatment: cells were starved overnight and then treated for 20 min (Insulin) at 100 ng/ml.

      Phosphatase treatment: membrane strips were incubated with 200 ul of phosphatase (150 U/ml) at 37 degrees for 1 hour.

    • All lanes : Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) at 1/1000 dilution

      Lane 1 : NIH/3T3 cell lysate
      Lane 2 : NIH/3T3 cell lysate
      Lane 3 : NIH/3T3 cell lysate


      Predicted band size : 56 kDa

      PDGF treatment: cells were starved overnight and then treated for 1 h with PDGF at 100 ng/ml.

      Phosphatase treatment: membrane strips were incubated with 200 ul of phosphatase (150 U/ml) at 37 degrees for 1 hour.

    • NIH3T3 cells starved overnight and treated with PDGF 50ng/mL for 1 hour (A) or vehicle (B). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton-X100. Primary antibody ab81283 was used at 1:4000 dilution and secondary antibody Dylight GAR594 (ab96897) at 1:1000 dilution.



    • Predicted band size : 56 kDa
      Primary : All Lanes : Anti AKT1 (phospho S473) antibody (ab81283) at 1:5000 dilution. Lane 1 = AKT1 (His tag) full length recombinant protein ab62279 - 50ng. Lane 2 = NIH3T3 serum starved overnight ? 15ug. Lane 3 = NIH3T3 serum starved overnight and treated with PDGF-AB 50ng/mL for 1 hour ? 15ug. Secondary : Lanes 1-3 : Goat polyclonal to Rabbit IgG ? H&L ? Pre-Adsorbed (HRP) at 1:5000 developed using the ECL technique. Performed under reducing conditions (50mM DTT ? Sample heated at 60?C). Predicted band size : 56kDa. Observed band size : 56kDa. Blocking step: 5% Milk in 50mM Tris+0.05% Tween for 1 hour at RT. Primary antibody buffer: 5% BSA in 50mM Tris+0.05% Tween overnight. Secondary antibody buffer: 5% Milk in 50mM Tris+0.05% Tween for 2 hours at RT. Exposure time : 5 minutes
    • NIH3T3 cells were starved overnight and treated with PDGF 50ng/mL or vehicle control for 1 hour prior to fixation with 4% paraformaldehyde. Levels of total Akt were measured using antibody ab81283 on an infrared in cell ELISA assay platform.
    • ab81283 staining AKT1 (phospho S473) in PC12 cells treated with galanin (1-29) (rat, mouse) (ab141153), by ICC/IF. Increase of AKT1 (phospho S473) expression correlates with increased concentration of galanin (1-29) (rat, mouse), as described in literature.
      The cells were incubated at 37°C for 24h in media containing different concentrations of ab141153 (galanin (1-29) (rat, mouse)) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab81283 (1/100) dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

    • Developed using the ECL technique

      Performed under reducing conditions.

      Predicted band size : 56 kDa
      Observed band size : 60 kDa (why is the actual band size different from the predicted?)


      Exposure time : 20 minutes

      MCF7 cells were incubated at 37°C for 2 hours with vehicle control (0 μM) and different concentrations of CCCP (ab 141229). Increased expression of AKT1 (phospho S473) (ab81283) in MCF7 cells correlates with an increase in CCCP concentration, as described in literature.

      Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated withab81283at 2 μg/ml andab8227at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 and visualised using ECL development solution.

    • ab81283 staining AKT1 (phospho S473) in PC3 cells treated with CAY10626 (ab120903), by ICC/IF. Decrease of AKT1 (phospho S473) expression correlates with increased concentration of CAY10626, as described in literature.
      The cells were incubated at 37°C for 24h in media containing different concentrations of ab120903 (CAY10626) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab81283 (1/100) dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
    • ab81283 staining AKT1 (phospho S473) in MCF7 cells treated with DAPT (ab120633), by ICC/IF. Decrease in expression of AKT1 (phospho S473) correlates with increased concentration of DAPT, as described in literature.
      The cells were incubated at 37°C for 24h in media containing different concentrations of ab120633 (DAPT) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab81283 (1/200 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

    • Developed using the ECL technique

      Performed under reducing conditions.

      Predicted band size : 56 kDa
      Observed band size : 60 kDa (why is the actual band size different from the predicted?)


      Exposure time : 20 minutes

      PC12 cells were incubated at 37°C for 24 hours with vehicle control (0 nM) and 1 μM of Galanin (1-15) (porcine, rat) (ab 141152). Decreased expression of AKT1 (phospho S473) (ab81283) in PC12 cells correlates with an increase in Galanin (1-15) (porcine, rat) concentration, as described in literature.

       Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 30μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated withab81283at 1 μg/ml andab8227at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 and visualised using ECL development solution.

    • ab81283 staining AKT1 (phospho S473) in Human peritoneal tumor tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (1/500) for 2 hours. An Alexa Fluor® 647-conjugated Donkey anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.

      See Abreview

    Anti-AKT1 (phospho S473) antibody [EP2109Y] (ab81283) 使用論文

    This product has been referenced in:
    • Chen Y  et al. FGFR antagonist induces protective autophagy in FGFR1-amplified breast cancer cell. Biochem Biophys Res Commun N/A:N/A (2016). WB ; Human . Read more (PubMed: 26993162) »
    • Liang D  et al. Therapeutic efficacy of apelin on transplanted mesenchymal stem cells in hindlimb ischemic mice via regulation of autophagy. Sci Rep 6:21914 (2016). WB ; Mouse . Read more (PubMed: 26902855) »

    See all 74 Publications for this product

    Product Wall

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Human Cell lysate - whole cell (HEK 293 cells)
    Gel Running Conditions Reduced Denaturing (10)
    Loading amount 20 µg
    Specification HEK 293 cells
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Username

    Abcam user community

    Verified customer

    投稿 Sep 23 2016

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Human Cell lysate - whole cell (Epithelial OVCA cell line)
    Gel Running Conditions Reduced Denaturing (10%)
    Loading amount 100 µg
    Treatment 0- 200µM Perifosine 24 hr
    Specification Epithelial OVCA cell line
    Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Username

    Abcam user community

    Verified customer

    投稿 Jul 13 2016

    Application Immunocytochemistry/ Immunofluorescence
    Sample Mouse Cell (Peritonei tumor)
    Permeabilization Yes - 0.3%Triton X 100
    Specification Peritonei tumor
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
    Fixative Paraformaldehyde
    Username

    Abcam user community

    Verified customer

    投稿 Jan 19 2016

    Application Western blot
    Loading amount 30 µg
    Gel Running Conditions Reduced Denaturing
    Sample Human Cell lysate - whole cell (macrophage)
    Specification macrophage
    Treatment 0.1 ug/ml LPS 15 min
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

    Abcam user community

    Verified customer

    投稿 Dec 12 2014

    Application Immunohistochemistry (Frozen sections)
    Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
    Sample Human Cell (Peritoneal tumor)
    Specification Peritoneal tumor
    Permeabilization No
    Fixative Paraformaldehyde
    Username

    Abcam user community

    Verified customer

    投稿 Oct 14 2014

    Application Western blot
    Loading amount 20 µg
    Gel Running Conditions Reduced Denaturing (4-12%)
    Sample Human Cell lysate - whole cell (A549)
    Specification A549
    Treatment Serum starvation followed by serum addition together with 100ng/ml of EGF for 0 to 3hrs
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

    Abcam user community

    Verified customer

    投稿 Mar 21 2014

    Thank you for contacting us.


    The exact epitope is proprietary, but the immunogen sequence is approximately 20 amino acids long and maps within amino acids 400-440 of p70 S6 Kinase of human origin.Thismeans, that both isoforms will be de...

    Read More

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