Recombinant Human Active Akt1 Protein, CF

(3 citations)
(1 Review)
  
  • Purity
    >70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
  • Activity
    The activity of Akt1 is typically 103-139 nmol/min/mg using a synthetic peptide substrate (RPRAATF) (see Activity Assay Protocol).
  • Source
    Spodoptera frugiperda, Sf 9 (baculovirus)-derived Accession # NM_005163
  • Accession #
  • N-terminal Sequence
    Analysis
    Using an N terminal GST tag
  • SDS-PAGE
    85 kDa
1775-KS
 
Formulation Supplied in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.25 mM DTT, 10 mM glutathione, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: This product is stable at ≤ ‑70° C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.
Assay Procedure
Materials
  • Active Kinase - Active Akt1 (0.1 μg/μL) diluted with Kinase Dilution Buffer and assayed as outlined in Figure 2. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer I, pH 7.2 - 25 mM MOPS, 12.5 mM beta -glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
  • Kinase Dilution Buffer, pH 7.2 - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold dilution) with 5% glycerol solution.
  • 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer I.
  • [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [33P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer I.
  • Substrate - Akt-sub synthetic peptide substrate (RPRAATF) diluted in distilled or deionized water to a final concentration of 1 mg/mL.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active Akt1, Kinase Assay Buffer I, Substrate, and Kinase Dilution Buffer on ice.
  3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL.
    a. Diluted Active Akt1: 10 μL
    b. Substrate (1 mg/mL Stock Solution): 5 μL
    c Distilled water (2-8 °C): 5 μL
  4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
  5. Initiate the reaction by the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
  6. After the 15 minute incubation period, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
  7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10 mL of phosphoric acid and make a 1 liter solution with distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
  8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
  9. Determine the corrected cpm by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below:


    Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
    Specific Activity (SA) = cpm for 5 μL [33P]-ATP/pmol of ATP (in 5 μL of a 250 μM ATP stock solution; i.e. 1250 pmol

    Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]
Data Images
The approximate molecular weight is 85 kDa and the average purity is 90%.
Background: Akt1

Akt1, also known as PKB alpha, is a serine/threonine kinase that belongs to the Akt family. Akt1 is activated in cells in response to diverse stimuli such as hormones, growth factors and extracellular matrix components and is involved in glucose metabolism, transcription, survival, cell proliferation, angiogenesis, and cell motility (1). Akt1 is frequently over-expressed and active in many types of human cancers including cancers of colon, breast, brain, pancreas, and prostate as well as lymphomas and leukemias (2).

  • References:
    1. Coffer, P.J. et al. (1998) Biochem J. 335:1.2.
    2. Anderson, K.E. et al. (1998) Curr. Biol. 8:684.
  • Long Name:
    v-Akt Murine Thymoma Viral Oncogene Homolog 1
  • Entrez Gene IDs:
    207 (Human); 11651 (Mouse); 25233 (Rat)
  • Alternate Names:
    AKT; Akt1; EC 2.7.11; EC 2.7.11.1; PKB alpha; PKBMGC99656; PRKBA; Protein kinase B; Proto-oncogene c-Akt; rac protein kinase alpha; RAC-alpha serine/threonine-protein kinase; RAC-alpha; RAC-PK-alpha; RACPKB-ALPHA; v-akt murine thymoma viral oncogene homolog 1
Related Research Areas
Citations:

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

3 Citations: Showing 1 - 3
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Species
Applications
Sample Type
  1. UBE2S, a novel substrate of Akt1, associates with Ku70 and regulates DNA repair and glioblastoma multiforme resistance to chemotherapy
    Oncogene, 2016;0(0):.
    Species: Human
    Sample Type: Protein
    Application: Enzyme Assay
  2. The PI3K/Akt signal hyperactivates Eya1 via the SUMOylation pathway.
    Authors: Sun Y, Kaneko S, Li X, Li X
    Oncogene, 2015;34(19):2527-37.
    Species: Human
    Sample Type: Cell Lysates
    Application: Enzyme Assay
  3. Dithiolethione compounds inhibit Akt signaling in human breast and lung cancer cells by increasing PP2A activity.
    Authors: Switzer CH, Ridnour LA, Cheng RY, Sparatore A, Del Soldato P, Moody TW, Vitek MP, Roberts DD, Wink DA
    Oncogene, 2009;28(43):3837-46.
    Species: Human
    Sample Type: Cell Lysates
    Application: Bioassay
Average Rating: 5 (Based on 1 review)

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