Recombinant Human Active PKC delta Protein, CF

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  • Purity
    >70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
  • Activity
    The activity of PKCδ is typically 249-337 nmol/min/mg using a synthetic peptide substrate (see Activity Assay Protocol).
  • Source
    Spodoptera frugiperda, Sf 9 (baculovirus)-derived
  • Accession #
  • N-terminal Sequence
    Using an N-terminal GST tag
    104 kDa
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, 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 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
  • Active Kinase - PKCδ (0.1 μg/μL) diluted with Kinase Dilution Buffer. 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 5-fold with 50 ng/mL 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.
  • PKC Lipid Activator - 0.5 mg/mL phosphatidylserine and 0.05 mg/mL diacylglycerol in 20 mM MOPS, pH 7.2, containing 1 mM CaCl2. Sonicate the lipid for 1 minute prior to use.
  • Substrate - CREBtide synthetic peptide substrate (KRREILSRRPSYR) 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 PKCδ, 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 PKCδ: 10 μL
    b. Substrate (1 mg/mL; on ice): 5 μL
    c. LPKC Lipid Activator: 2.5 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 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)]
Data Images
The approximate molecular weight is 104 kDa and the average purity is 90%.
Background: PKC delta

Protein Kinase C delta (PKCδ) is a member of the protein kinase C (PKC) family of serine-threonine kinases. It is a 104 kDa protein kinase that shows strict dependence on the presence of phospholipids but shows no activation by Ca2+ (1). Phosphatidylinositol is the most potent activator of PKCδ. Northern blot analysis indicates that PKCδ is widely distributed in almost all the tissues and is a major isoform of PKC expressed in hemopoietic cells (2). PKCδ is involved in fundamental cellular functions regulated by diacylglycerols and mimicked by phorbol esters.

  • References:
    1. Leibersperger, H. et al. (1991) J. Biol. Chem. 266:14778.
    2. Mischak, H. et al. (1991) Biochemistry 30:7925.
  • Long Name:
    Protein Kinase C delta
  • Entrez Gene IDs:
    5580 (Human)
  • Alternate Names:
    EC 2.7.11; EC; MAY1; MGC49908; nPKC-delta; PKCD; PRKCD; protein kinase C delta type; protein kinase C delta VIII; protein kinase C, delta
Related Research Areas

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.

Showing Results 1 - 1 of 1

  1. Phosphorylation of NLRC4 is critical for inflammasome activation.
    Authors: Qu Y, Misaghi S, Izrael-Tomasevic A, Newton K, Gilmour L, Lamkanfi M, Louie S, Kayagaki N, Liu J, Komuves L, Cupp J, Arnott D, Monack D, Dixit V
    Nature, 2012;490(7421):539-42.
    Species: Mouse
    Sample Type: Whole Cells
    Application: Bioassay

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