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Purified anti-AKT Phospho (Ser473) Antibody

Pricing & Availability
Clone
A21001C (See other available formats)
Regulatory Status
RUO
Other Names
Protein kinase B alpha, PRK-BA, PKB, Serine/Threonine specific kinase RAC alpha, Protein kinase Akt, RAC
Isotype
Mouse IgG1, κ
Ave. Rating
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Product Citations
publications
A21001C_PURE_Akt_Antibody_080222
Whole cell extracts (15 µg total protein) from serum-starved HeLa cells untreated (-) or treated (+) with IGF-1 (100 ng/ml, 30 min), and from serum-starved NIH/3T3 cells untreated (-) or treated (+) with PDGF-BB (200 ng/mL, 15 min) were resolved on a 4-12% Bis-Tris gel, transferred to a PVDF membrane, and probed with 1 µg/mL (1:500 dilution) of purified anti-AKT Phospho (Ser473) (clone A21001C) overnight at 4°C. Proteins were visualized by chemiluminescence detection using HRP goat anti-mouse IgG (Cat. No. 405306) at a 1:3000 dilution. Equal AKT Phospho (Ser473) loading was confirmed by probing membranes with anti-pan AKT1 (clone O94E10) (Cat. No. 680302) at a 1:500 dilution. Direct-Blot™ HRP anti-GAPDH (Cat. No. 607904) was used as a loading control at a 1:50000 dilution. Western-Ready™ ECL Substrate Plus Kit (Cat. No. 426317) was used as a detection agent. Lane M: Molecular weight marker
  • A21001C_PURE_Akt_Antibody_080222
    Whole cell extracts (15 µg total protein) from serum-starved HeLa cells untreated (-) or treated (+) with IGF-1 (100 ng/ml, 30 min), and from serum-starved NIH/3T3 cells untreated (-) or treated (+) with PDGF-BB (200 ng/mL, 15 min) were resolved on a 4-12% Bis-Tris gel, transferred to a PVDF membrane, and probed with 1 µg/mL (1:500 dilution) of purified anti-AKT Phospho (Ser473) (clone A21001C) overnight at 4°C. Proteins were visualized by chemiluminescence detection using HRP goat anti-mouse IgG (Cat. No. 405306) at a 1:3000 dilution. Equal AKT Phospho (Ser473) loading was confirmed by probing membranes with anti-pan AKT1 (clone O94E10) (Cat. No. 680302) at a 1:500 dilution. Direct-Blot™ HRP anti-GAPDH (Cat. No. 607904) was used as a loading control at a 1:50000 dilution. Western-Ready™ ECL Substrate Plus Kit (Cat. No. 426317) was used as a detection agent. Lane M: Molecular weight marker
  • A21001C_PURE_Akt_Antibody_2_080222
    Untreated Jurkat cells (positive control, panel A) and Jurkat cells treated with 1 µM wortmannin for 2 hours (negative control, panel B) were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with ice cold MeOH for 10 minutes, and blocked with 5% FBS for 60 minutes. Cells were then intracellularly stained with 5.0 µg/mL purified anti-AKT Phospho (Ser473) (clone A21001C) overnight at 4°C, followed by incubation with Alexa Fluor® 594 goat anti-mouse IgG (Cat. No. 405326) at 2.0 µg/mL. Nuclei were counterstained with DAPI and the image was captured with a 60X objective. Scale: 50 µm
  • A21001C_PURE_Akt_Antibody_3_080222
    Jurkat cells untreated (positive target, filled histogram), and treated with 1.0 µM wortmannin for 2 hours (negative target, solid line, open histogram) were fixed and permeabilized using the True-Phos™ Fix/Perm Buffer Set (Cat. No. 425401) and intracellularly stained with either purified anti-AKT Phospho (Ser473) (clone A21001C) (solid line histograms) or purified mouse IgG1, κ isotype control (dashed line, open histogram) followed by PE goat anti-mouse IgG.
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606551 25 µg 118 CHF
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606552 100 µg 347 CHF
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Description

AKT (also known as protein kinase B alpha) is a 60 kD serine/threonine-specific kinase containing a pleckstrin domain. AKT plays a critical role in controlling survival and apoptosis. This kinase is ubiquitously expressed and translocates to the membrane upon cell activation. It is activated by insulin and various growth and survival factors to function in a wortmannin-sensitive pathway involving PI 3-kinase. AKT is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2, responsible for phosphorylation of AKT at Ser473, has been identified as a mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1. Activated AKT phosphorylates a wide range of substrates including transcription factors (e.g. FOXO1), kinases (GSK-3beta, Raf-1, ASK, Chk1) and other proteins with important signaling roles (e.g. Bad, MDM2). AKT promotes cell survival by inhibiting apoptosis through phosphorylation of several targets, including Bad, forkhead transcription factor, c-Raf and caspase-9. AKT also plays a critical role in cell growth by directly phosphorylating mTOR in a rapamycin-sensitive complex containing raptor. More importantly, AKT phosphorylates and inactivates tuberin (TSC2), an inhibitor of mTOR within the mTOR-raptor complex. 

Product Details
Technical Data Sheet (pdf)

Product Details

Verified Reactivity
Human, Mouse
Antibody Type
Monoclonal
Host Species
Mouse
Immunogen
Synthetic peptide of human AKT phosphorylated at Ser473
Formulation
Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide
Preparation
The antibody was purified by affinity chromatography.
Concentration
0.5 mg/mL
Storage & Handling
The antibody solution should be stored undiluted between 2°C and 8°C.
Application

WB - Quality tested
ICC, ICFC - Verified
Recommended Usage

Each lot of this antibody is quality control tested by western blotting. For western blotting, the suggested use of this reagent is 0.25 - 1.0 µg/mL. For immunocytochemistry, a concentration range of 2.5 - 5.0 μg/mL is recommended. For flow cytometric staining, the suggested use of this reagent is ≤ 0.03 µg per million cells in 100 µL volume. It is recommended that the reagent be titrated for optimal performance for each application.

Application Notes

This clone was tested by Western Blot using lysates prepared from HeLa cells untreated or treated with IGF-1, and NIH/3T3 cell untreated or treated with PDGF. 

This clone is suitable for ICC in cells treated with PFA and permeabilized with either methanol or Triton X-100.

RRID
AB_2924618 (BioLegend Cat. No. 606551)
AB_2924618 (BioLegend Cat. No. 606552)

Antigen Details

Structure
AKT is a 480 amino acid protein with a predicted molecular weight of 56 kD
Distribution

Ubiquitously expressed, translocates to the membrane upon activation

Function
Catalytically inactive multimeric complex. AKT is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 and by phosphorylation within the carboxy terminus at Ser473 by PDK2.
Interaction
Interacts through pleckstrin homology domain with second messengers. Interacts with AKTIP, CDKN1B.
Biology Area
Cell Biology, Cell Cycle/DNA Replication, Cell Death
Molecular Family
Phospho-Proteins, Protein Kinases/Phosphatase
Antigen References
  1. Jacinto E, et al. 2006. Cell. 127:125-37.
  2. Inoki K, et al. 2002. Nat Cell Biol. 4:648-57.
  3. Franke TF, et al. 1995. Cell. 81:727-36.
  4. Brunet A, et al. 1999. Cell. 96:857-68.
  5. Yang WL, et al. 2010. Cell Cycle. 9:487-97.
Gene ID
207 View all products for this Gene ID
UniProt
View information about AKT Phospho Ser473 on UniProt.org
Go To Top Version: 1    Revision Date: 08.02.2022

For Research Use Only. Not for diagnostic or therapeutic use.

 

This product is supplied subject to the terms and conditions, including the limited license, located at www.biolegend.com/terms) ("Terms") and may be used only as provided in the Terms. Without limiting the foregoing, BioLegend products may not be used for any Commercial Purpose as defined in the Terms, resold in any form, used in manufacturing, or reverse engineered, sequenced, or otherwise studied or used to learn its design or composition without express written approval of BioLegend. Regardless of the information given in this document, user is solely responsible for determining any license requirements necessary for user’s intended use and assumes all risk and liability arising from use of the product. BioLegend is not responsible for patent infringement or any other risks or liabilities whatsoever resulting from the use of its products.

 

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This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
If you need assistance with selecting the best format contact our expert technical support team.

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