Recombinant Human Active PDK-1 Protein, CF

Catalog # Availability Size / Price Qty
864-KS-010
Recombinant Human Active PDK-1 Protein SDS-PAGE
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Recombinant Human Active PDK-1 Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Activity
The specific activity of PDK-1 was determined to be 102 nmol/min/mg using a synthetic peptide substrate.
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human PDK-1 protein
Accession #
N-terminal Sequence
Analysis
Using an N-terminal His tag
SDS-PAGE
67 kDa

Product Datasheets

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864-KS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

864-KS

Formulation Supplied in 50 mM sodium phosphate (pH 7.0), 300 mM NaCl, 0.25 mM DTT, 150 mM imidazole, 0.1 mM PMSF, and 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 one 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 PDK-1 (0.1 μg/μL) diluted with Kinase Dilution Buffer III. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer I - 25 mM MOPS, pH 7.2, 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 III - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold) with 50 ng/μL BSA 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. Store 200 μL aliquots at ≤ -20 °C.
  • [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. Store 1 mL aliquots at ≤ -20° C.
  • Substrate - PDKtide synthetic peptide substrate (KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC) diluted in distilled or deionized water to a final concentration of 1.0 mg/mL.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active PDK-1, Kinase Assay Buffer I, Substrate, and Kinase Dilution Buffer III 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 PDK-1: 10 μL
    b. Substrate (1.0 mg/mL; on ice): 5 μL
    c. Distilled or deionized water: 5.0 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 with 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, 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 (add 10 mL of phosphoric acid to 990 mL of 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 subtracting 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)]

Scientific Data

SDS-PAGE Recombinant Human Active PDK-1 Protein SDS-PAGE View Larger

The approximate molecular weight is 67 kDa and the purity is > 95%.

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Background: PDK-1

PDK-1 (3-phosphoinositide-dependent protein kinase) is activated by the presence of PtdIns(3,4,5)P3 or PtdIns(3,4)P2 (1). PDK‑1 then activates protein kinase B (PKB) that, in turn, inactivates glycogen synthase kinase-3 (GSK-3). The phosphorylation of other proteins by PKB and GSK-3 is likely to mediate many of the intracellular actions of insulin. Thus, PDK‑1 plays a key role in mediating many of the actions of the second messenger(s) PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2. Human PDK‑1 is a 556 amino acid residue monomeric enzyme comprised of a catalytic domain that is most similar to the PKA, PKB, and PKC subfamily of protein kinases.

References
  1. Cohen, P. et al. (1997) FEBS Letter 410:3.
  2. Alessi, D.R. et al. (1997) Current Biol. 7:261.
Long Name
Phosphoinositide Dependent Kinase-1
Entrez Gene IDs
5170 (Human); 18607 (Mouse); 81745 (Rat)
Alternate Names
3-phosphoinositide-dependent protein kinase 1; 3-phosphoinositide-dependent protein kinase 2 pseudogene; PDK1; PDK-1; PDPK1; PDPK2; PDPK2P; PkB kinase like gene 1; PKB kinase

Citation for Recombinant Human Active PDK-1 Protein, CF

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.

1 Citation: Showing 1 - 1

  1. The Akt activation inhibitor TCN-P inhibits Akt phosphorylation by binding to the PH domain of Akt and blocking its recruitment to the plasma membrane.
    Authors: Berndt N, Yang H, Trinczek B, Betzi S, Zhang Z, Wu B, Lawrence NJ, Pellecchia M, Schonbrunn E, Cheng JQ, Sebti SM
    Cell Death Differ., 2010-05-21;17(11):1795-804.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Bioassay

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