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Recombinant Human Active Akt1 Protein, CF

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The approximate molecular weight is 85 kDa and the average purity is 90%.
The approximate molecular weight is 85 kDa and the average purity is 90%.

Product Details

Summary
Reactivity HuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

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Recombinant Human Active Akt1 Protein, CF Summary

Details of Functionality
The specific activity of Akt1 was determined to be 189 nmol/min/mg as per activity assay protocol.
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human Akt1 protein
Accession #
N-terminal Sequence
Using an N terminal GST tag
Protein/Peptide Type
Recombinant Proteins
Gene
AKT1
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Applications/Dilutions

Dilutions
  • Bioactivity
SDS-PAGE
85 kDa
Publications
Read Publications using
1775-KS in the following applications:

Packaging, Storage & Formulations

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.
Buffer
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.
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Assay Procedure
  • Active Kinase - Active Akt1 (0.1 μg/μL) diluted with Kinase Dilution Buffer and assayed as outlined in sample activity plot. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer III (5X) - 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2, and 0.5 mg/mL BSA. Add fresh DTT prior to use to a concentration of 50 μM.
  • Kinase Dilution Buffer IX (1X) - Kinase Assay Buffer III diluted at a 1:4 ratio (5x dilution) with cold water. Add fresh DTT to the aliquot prior to use to a final concentration of 50 μM.
  • ADP-Glo™ Kinase Assay Kit - ATP solution 10 mM, ADP solution 10 mM, ADP-Glo Reagent, Kinase Detection Reagent
  • Substrate - Akt (PKB) synthetic peptide substrate (CKRPRAASFAE) diluted in 25 mM Tris-HCl Buffer (pH 7.5) to a final concentration of 1 mg/mL.
  1. Thaw the Active Akt1, Kinase Assay Buffer III (5X), and Substrate on ice. Prepare a 15 μL enzyme dilution at the desired concentration, with Kinase Dilution Buffer IX (1X) in a pre-chilled 96-well plate.
  2. Prepare a Substrate/ATP mixture as follows (25μM example):
    a. 10 μM ATP solution: 1 μL
    b. Kinase Assay Buffer III (5X): 79 μL
    c. Substrate at 1 mg/mL: 80 μL
  3. Transfer the following reaction components prepared in step 2 to a 384-well opaque plate bringing the reaction volume up to 5 μL:
    a. 3 μL of diluted Active Akt1
    b. 2 μL of Substrate/ATP mix as prepared in step 2. This initiates the reaction.
  4. Set up the blank control as outlined in step 2, excluding the addition of the kinase. Replace the kinase with an equal volume of Kinase Dilution Buffer IX (1x).
  5. Incubate at ambient temperature for 40 minutes.
  6. After the 40 minute incubation period, terminate the reaction and deplete the remaining ATP by adding 5 μL of ADP-Glo™ Reagent. Spin down and shake the 384-well plate. Incubate the reaction mixture for another 40 minutes at ambient temperature.
  7. Add 10 μL of the Kinase Detection Reagent to the 384-well plate and incubate the reaction mixture for another 40 minutes at ambient temperature.
  8. Read the 384-well plate using the Luminescence Module Protocol on a GloMax®-Multi Microplate Multimode Reader.
  9. Determine the corrected activity (RLU) by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below.


    Calculation of Specific Activity of ADP (RLU/pmol)
    From ADP standard curve, determine RLU/pmol of ADP

    Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)]

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active Akt1 Protein, CF

  • AKT serine/threonine kinase 1
  • AKT
  • Akt1
  • AKT1m
  • CWS6
  • EC 2.7.11
  • EC 2.7.11.1
  • PKB alpha
  • PKB
  • PKBMGC99656
  • PRKBA
  • Protein Kinase B Alpha
  • Protein kinase B
  • Proto-oncogene c-Akt
  • rac protein kinase alpha
  • RAC
  • RAC-alpha serine/threonine-protein kinase
  • RAC-alpha
  • RAC-PK-alpha
  • RACPKB-ALPHA
  • Serine-Threonine Protein Kinase
  • v-akt murine thymoma viral oncogene homolog 1
  • V-Akt Murine Thymoma Viral Oncogene-Like Protein 1

Background

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).

  1. Coffer, P.J. et al. (1998) Biochem J. 335:1.2.
  2. Anderson, K.E. et al. (1998) Curr. Biol. 8:684.

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Publications for AKT1 (1775-KS)(4)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 3 applications: Bioassay, Enzyme Assay, Immunoprecipitation.


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Bioassay
(1)
Enzyme Assay
(2)
Immunoprecipitation
(1)
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Human
(4)
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Reviews for AKT1 (1775-KS) (0)

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FAQs for AKT1 (1775-KS). (Showing 1 - 5 of 5 FAQs).

  1. How do I choose secondary antibodies to label the same cells when I have two primary antibodies from the same host?
    • Use isotype-specific secondary antibodies if the primary antibodies are of different isotypes. You can also make direct conjugates of the primary antibodies by use of antibody labeling kits, dyes, or custom conjugations (please contact Technical Support for custom orders).
  2. Why are many of your antibodies formulated with sodium azide and BSA?
    • Sodium azide is a preservative which is added to prevent bacterial growth. BSA is added as a protein stabilizer.
  3. Do your HRP-conjugated antibodies contain sodium azide?
    • No. None of our HRP-conjugated antibodies contain sodium azide as this agent inhibits the activity of HRP.
  4. I am looking for a antibody that recognizes human Akt1 but NOT Akt2 or 3, for Western blot analyses. I also want that antibody to recognize Akt1 regardless of its phosphorylated form.
    • At the moment we do not have an AKT1 antibody that definitively does not react with either AKT2 or AKT3.
  5. What is the molecular weight of your antibodies?
    • All IgG antibodies are approximately 150 kDa (each heavy chain is about 50 kDa and each light chain is about 25 kDa).

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Bioinformatics

Gene Symbol AKT1
Uniprot