Recombinant Human Active PKC delta Protein, CF

R&D Systems | Catalog # 4585-KS

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Key Product Details

  • R&D Systems Sf 9 (baculovirus)-derived Recombinant Human Active PKC delta Protein (4585-KS)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

Sf 9 (baculovirus)

Accession Number

NM_006254

Applications

Bioactivity
View all PKC delta Proteins and Enzymes »

Product Specifications

Source

Spodoptera frugiperda, Sf 9 (baculovirus)-derived human PKC delta protein

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

N-terminal Sequence Analysis

Using an N-terminal GST tag

SDS-PAGE

104 kDa

Activity

The specific activity of PKC delta was determined to be 185 nmol/min/mg using a synthetic peptide substrate.

Scientific Data Images for Recombinant Human Active PKC delta Protein, CF

Recombinant Human Active PKC delta Protein SDS-PAGE

Recombinant Human Active PKC delta Protein SDS-PAGE.

The approximate molecular weight is 104 kDa and the purity is > 90%.

Formulation, Preparation, and Storage

4585-KS
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.

Background: PKC delta

Protein Kinase C delta (PKC delta ) 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 PKC delta. Northern blot analysis indicates that PKC delta is widely distributed in almost all the tissues and is a major isoform of PKC expressed in hemopoietic cells (2). PKC delta 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

Alternate Names

PRKCD

Entrez Gene IDs

5580 (Human)

Gene Symbol

PRKCD

UniProt

NM_006254 (Human)

Additional PKC delta Products

Product Documents for Recombinant Human Active PKC delta Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

Product Specific Notices for Recombinant Human Active PKC delta Protein, CF

For research use only

Citations for Recombinant Human Active PKC delta Protein, CF

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Protocols

View specific protocols for Recombinant Human Active PKC delta Protein, CF (4585-KS):

Materials
  • Active Kinase - Active PKC delta (0.05 μg/μL) diluted with Kinase Dilution Buffer IX (1X) and assayed as outlined in sample activity plot. Note: These are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active PKC delta for optimal results.
  • 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 final concentration of 250 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 - 10 mM of ATP Solution, 10 mM of ADP Solution, ADP-Glo™ Reagent, and Kinase Detection Reagent.
  • Substrate - CREBtide peptide substrate was diluted in distilled water to a final concentration of 1 mg/mL.
  • PKC Lipid Activator 10X - pH 7.2, 10X stock solution containing 0.5 mg/mL phosphatidylserine, 100 μg/mL diacylglycerols, 0.15% Triton X-100, 1 mM DTT, 2 mM CaCl2, 20 mM MOPS.
  1. Thaw the Active PKC delta, 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/Cofactor/ATP mixture as follows (25 μM ATP example):
    a. 2.5 mM ATP Solution: 2 μL
    b. Substrate at 1 mg/mL: 40 μL
    c. Kinase Assay Buffer III (5X): 58 μL
    d. PKC Lipid Activator (10X): 20 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. 2 μL of diluted Active PKC delta
    b. 3 μL of Substrate/Cofactor/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. Then 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 30 minutes at ambient temperature.
  8. Read the 384-well reaction 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/min/μg or nmol/min/mg)
    Corrected RLU from reaction / [(SA of ADP in RLU/pmol) x (Reaction time in min) x (Enzyme amount in μg or mg)]

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