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.
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.
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
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.
Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
Thaw the Active Akt1, Kinase Assay Buffer I, Substrate, and Kinase Dilution Buffer on ice.
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
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.
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.
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.
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.
Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
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)]
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
Protein Kinase B Alpha
Protein kinase B
rac protein kinase alpha
RAC-alpha serine/threonine-protein kinase
Serine-Threonine Protein Kinase
v-akt murine thymoma viral oncogene homolog 1
V-Akt Murine Thymoma Viral Oncogene-Like Protein 1
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).
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FAQs for AKT1 (1775-KS). (Showing 1 - 5 of 5 FAQs).
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).
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.
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.
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.
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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PRODUCT AVAILABILITY: Update Regarding the Evolving COVID-19 Situation
Bio-Techne appreciates the critical role that you and our products and services play in research efforts to further scientific innovation and discovery. We are continually assessing our manufacturing and supplier capabilities during the COVID-19 situation and are implementing precautionary measures to ensure uninterrupted supply of products and services. Currently, and as we abide by local shelter in place orders across the world, we are fully operational and do not anticipate any material supply disruptions across our Bio-Techne brands and product lines. As the situation evolves, our goal is to utilize preventive measures to reduce the threat that COVID-19 poses to our ability to meet the needs of our customers globally.