Recombinant Human Active Akt1 Protein, CF

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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 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 human Akt1 protein
Accession # NM_005163
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

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 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)]

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
  • 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

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)(3)

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

  1. 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.

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Blogs on AKT1. Showing 1-10 of 11 blog posts - Show all blog posts.

Using a STAT3 antibody in chromatin immunoprecipitation (ChIP)
Signal transducer and activator of transcription 3 (STAT3) is an important oncogenic transcriptional factor that mediates tumor induced immune suppression.  Specifically, STAT3 transmits signals from cytokines and growth factor receptors in the pla...  Read full blog post.

Altered expression of BCL2 in cancer
Similar to other cell processes, the balance between cell survival and cell death is an important equilibrium that when altered expression of genes can lead to a variety of disease.  For example, too little cell death can promote cell overgrowth a...  Read full blog post.

AKT1 - Regulating cell growth and survival through phosphorylation
AKT1 is a serine/threonine protein kinase with homology to protein kinase A (PKA) and protein kinase C (PKC). AKT1 contains the central kinase domain sandwiched between a pleckstrin homology domain and a regulatory domain (1). AKT1 is regulated by...  Read full blog post.

Akt1 - a central player in cell survival signaling
Akt1 is one of three isoforms of Akt belonging to the AGC family of serine/threonine kinases (Akt1, Akt2, and Akt3). All Akt isoforms contain an N-terminal Plekstrin Homology (PH) domain, a C-terminal regulatory domain, and a central catalytic kin...  Read full blog post.

AKT1, Scene 1: The Cell Must Go On
Akt1 is a serine/threonine-specific protein kinase involved in many cellular signaling pathways. The major function of this kinase is to mediate cell survival, but it also plays key roles in various other cellular functions such as glycogen synthesis...  Read full blog post.

Caspase 9 and Mitochondrial Apoptosis Regulation
Caspase 9 (also termed ICE-LAP6, Mch6, Apaf-3) is a member of cysteine protease family of caspases and is encoded by the CASP9 gene in humans. Caspase-9 is involved in mitochondrial apoptosis pathway and is an initiator caspase. Pro-caspase-9 is activ...  Read full blog post.

Mapping Signal Transduction with mTOR Antibodies
The protein encoded by mTOR (mammalian target of rapamycin), also known as dTOR in Drosophila, belongs to a family of phosphatidylinositol kinase-related kinases. These kinases regulate fundamental processes of cell growth, proliferation, metabolism...  Read full blog post.

EZH2: Epigenetic Regulation Made Easy!
Enhancer of Zeste homolog 2 (EZH2) is the methyltransferase enzyme responsible for trimethylating lysine 27 on histone H3 to produce H3K27Me3. EZH2 is a polycomb group protein that is an essential epigenetic regulator that is often found deregulated i...  Read full blog post.

Antibodies Targeting the mTOR Pathway for Many Diseases
The mammalian target of rapamycin (mTOR) is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway that mediates cell survival and proliferation. It is increasingly apparent that mTOR signaling impac...  Read full blog post.

AKT Antibody Assays: A Complex Area with an Easy Solution
We at Novus Biologicals place a lot of emphasis on the kinase signaling pathways. Kinases, or phosphotransferase enzymes play a key role in phosphorylation signaling. Over 500 human protein kinases have so far been discovered. They play essential role...  Read full blog post.

Showing 1-10 of 11 blog posts - Show all blog posts.

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Bioinformatics

Gene Symbol AKT1
Uniprot