Recombinant Human Active PKC delta Protein, CF

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

• Reactivity: Hu
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant Human Active PKC delta Protein, CF Summary

• Details of Functionality: The specific activity of PKCδ was determined to be 185 nmol/min/mg using a synthetic peptide substrate.
• Source: Spodoptera frugiperda, Sf 9 (baculovirus)-derived human PKC delta protein
• Accession #: NM_006254
• N-terminal Sequence: Using an N-terminal GST tag
• Protein/Peptide Type: Recombinant Proteins
• Gene: PRKCD
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• SDS-PAGE: 104 kDa

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, and 25% Glycerol.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Active Kinase - Active PKCδ (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 PKCδ 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 PKCδ, 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 PKCδ
     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)]

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active PKC delta Protein, CF

• EC 2.7.11
• EC 2.7.11.13
• MAY1
• MGC49908
• nPKC-delta
• PKC delta
• PKCD
• PKCy
• PRKCD
• protein kinase C delta type
• protein kinase C delta VIII
• protein kinase C, delta

Background

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.

1. Leibersperger, H. et al. (1991) J. Biol. Chem. 266:14778.
2. Mischak, H. et al. (1991) Biochemistry 30:7925.

Publications for PKC delta (4585-KS)(1)

Publication using 4585-KS

• 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 Phosphorylation of NLRC4 is critical for inflammasome activation. Nature, 2012-08-12;490(7421):539-42. 2012-08-12 [PMID: 22885697] (Bioassay, Mouse)

Bioinformatics

• Gene Symbol: PRKCD
• Uniprot:
  • NM_006254(Human)