V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1) (pS473) antibody

Antigen: V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1)

Synonyms: AKT, PKB, RAC, PRKBA, MGC99656, PKB-ALPHA, RAC-ALPHA, Akt, pkb, xAct, akt-1, v-akt, v-akt1, AKT1, AKT/PKB, D-Akt, DAKT1, DAKT1/PKB, DPKB, DRAC-PK, DRAC-PK66; DRAC-PK85, Dakt, PKB/AKT, PKB/dAKT, RacPK, dAKT/dPKB, dAkt/PKB, l(3)04226, l(3)89Bq, DmelCG4006, CG4006

Epitope: pS473

Clonality: Monoclonal (17B6-B11-A12-C3-G7-G12)

Host: Mouse

Reactivity: Human

Application: ELISA, Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP)

Catalog no.: ABIN349614

Additional Information

Alternative name: AKT pS473

Gene ID: NP_005154

Swiss-Prot: P31749

Immunogen: This monoclonal antibody was produced by repeated immunizations with a synthetic peptide corresponding to residues surrounding S473 of human AKT1 protein.

Format: Liquid (sterile filtered)

Isotype: IgG1-kappa  (Matching secondary antibodies)

Clone: 17B6-B11-A12-C3-G7-G12

Description: AKT is a component of the PI-3 kinase pathway and is activated by phosphorylation at Ser 473 and Thr 308. AKT is a cytoplasmic protein also known as AKT1, Protein Kinase B (PKB) and rac (related to A and C kinases). AKT is a key regulator of many signal transduction pathways. AKT exhibits tight control over cell proliferation and cell viability. Overexpression or inappropriate activation of AKT is noted in many types of cancer. AKT mediates many of the downstream events of PI 3-kinase (a lipid kinase activated by growth factors, cytokines and insulin). PI 3-kinase recruits AKT to the membrane, where it is activated by PDK1 phosphorylation. Once phosphorylated, AKT dissociates from the membrane and phosphory-lates targets in the cytoplasm and the cell nucleus. AKT has two main roles: (i) inhibition of apoptosis, (ii) promotion of proliferation.

Specificity: This product was purified from concentrated tissue culture supernate by Protein A chromatography. This antibody is specific for human and mouse AKT protein phosphorylated at S473. A BLAST analysis was used to suggest cross-reactivity with AKT pS473 from human, mouse, rat and chimpanzee sources based on 100% homology with the immunizing sequence. Cross-reactivity with AKT from other sources has not been determined. Cross-reactivity with AKT2 and AKT3 has not been determined. Hybridoma : Produced by fusing BALB/c mouse splenocytes and mouse myeloma SP2/O cells using conventional technology.

Synonyms: AKT, PKB, RAC, PRKBA, MGC99656, PKB-ALPHA, RAC-ALPHA, Akt, pkb, xAct, akt-1, v-akt, v-akt1, AKT1, AKT/PKB, D-Akt, DAKT1, DAKT1/PKB, DPKB, DRAC-PK, DRAC-PK66; DRAC-PK85, Dakt, PKB/AKT, PKB/dAKT, RacPK, dAKT/dPKB, dAkt/PKB, l(3)04226, l(3)89Bq, DmelCG4006, CG4006

Application Details

Protocol: This monoclonal antibody is suitable for ELISA,immunohistochemistry, immunoprecipitation and western blotting. Expect a band approximately 56 kDa in size corresponding to phosphorylated AKT protein by western blotting in the appropriate cell lysate or extract. This phospho-specific monoclonal antibody reacts with human and mouse AKT pS473 and shows minimal reactivity by ELISA against the non-phosphorylated form of the immunizing peptide. Specific conditions for reactivity should be optimized by the end user. For immunohistochemistry use formalin-fixed paraffinembedded sections. No pre-treatment of sample is required. ELISA 1:20,000; WESTERN BLOT 1:500 - 1:3,000; IMMUNOHISTOCHEMISTRY 20 μg/ml; OTHER APPLICATIONS User Optimized

Application Notes: Recommended Dilutions: ELISA 1:20,000 WESTERN BLOT 1:500 - 1:3,000 IMMUNOHISTOCHEMISTRY 20 ug/ml OTHER APPLICATIONS User Optimized. Note: This monoclonal antibody is suitable for ELISA, immunohistochemistry, immunoprecipitation and western blotting. Expect a band approximately 56 kDa in size corresponding to phosphorylated AKT protein by western blotting in the appropriate cell lysate or extract. This phospho-specific monoclonal antibody reacts with human and mouse AKT pS473 and shows minimal reactivity by ELISA against the non-phosphorylated form of the immunizing peptide. Specific conditions for reactivity should be optimized by the end user. For immunohistochemistry use formalin-fixed paraffin-embedded sections. No pre-treatment of sample is required.

Concentration: 1.0 mg/ml (by UV absorbance at 280 nm)

Buffer: Stabilizer: None. Preservative: 0.01% (w/v) Sodium Azide. Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2.

Storage: Store vial at -20o C or below prior to opening. Dilute only prior to immediate use. For extended storage, aliquot contents and freeze at -20o C or below. Avoid cycles of freezing and thawing. Expiration date is one (1) year from date of opening.

Research Area: Apoptosis/Necrosis, Proliferation Markers

Restrictions: For Research Use only

Images

Western blot using Anti-AKT pS473 monoclonal antibody shows detection of phosphorylated AKT (indicated by arrowhead at ~56 kDa) on PDGF stimulated NIH/3T3 cell lysates (lane 2). No reactivity is seen for non-phosphorylated AKT in untreated cells (lane 1). Each lane contained approximately 10 ug of lysate. All samples were loaded on to a 4-20% gradient gel for separation. After electrophoresis, the gel was blocked with 5% BLOTTO (p/n B501-0500 in TBS for 90 min at RT. The membrane was probed with the primary antibody at a 1:10,000 dilution in TBS with 0.05% Tween-20 with 1% BSA, for 1 h at 4o C. For detection HRP conjugated Gt-a-Mouse IgG (p/n 610-103-121) was used at a 1:20,000 dilution for 1 h at 4o C with FemtoMaxTM enhanced chemiluminescent reagent (p/n FEMTOMAX-100). Images were captured using 2X2 binning for 10-20 sec using a BioSpectrum Imaging System (UVP Ltd.)

Immunohistochemistry using Anti-AKT pS473 monoclonal antibody shows detection of phosphory-lated AKT pS473 in human prostate tissue. The antibody was used at 20 ug/mL. The staining is much stronger than the weak basal level of phosphorylation in normal prostate tissue. Tissue was formalin fixed and paraffin embedded. No pre-treatment of sample was required. Signal was developed using Dako's Techmate streptavidin-biotin reagents. Personal communication, Glenna Burmer, Lifespan Biosciences, Seattle, WA.

anti-V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1) (pS473) antibody (Image 3)

Publications

Publications: Jones, Jakubowicz, Pitossi et al.: "Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 88, Issue 10, pp. 4171-5, 1991 (PubMed).

Staal: "Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, Issue 14, pp. 5034-7, 1987 (PubMed).

Alessi: "Discovery of PDK1, one of the missing links in insulin signal transduction. Colworth Medal Lecture." in: Biochemical Society transactions, Vol. 29, Issue Pt 2, pp. 1-14, 2001 (PubMed).

Lawlor, Alessi: "PKB/Akt: a key mediator of cell proliferation, survival and insulin responses?" in: Journal of cell science, Vol. 114, Issue Pt 16, pp. 2903-10, 2001 (PubMed).

Ito, Nishibori, Ito et al.: "mTORC1 activation triggers the unfolded protein response in podocytes and leads to nephrotic syndrome." in: Laboratory investigation; a journal of technical methods and pathology, 2011 (PubMed).