Recombinant Human Active PKA C alpha Protein, CF

The approximate molecular weight is 69 kDa and the average purity is 82%.

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

Recombinant Human Active PKA C alpha Protein, CF Summary

• Details of Functionality: The activity of PKA C alpha is typically 1626-2201 nmol/min/mg using a synthetic peptide substrate.
• Source: Spodoptera frugiperda, Sf 9 (baculovirus)-derived human PKA C alpha protein
• Accession #: NM_002730
• N-terminal Sequence:

  Using an N-terminal GST tag

• Protein/Peptide Type: Recombinant Proteins
• Gene: PRKACA
• 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: 69 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, 10 mM Glutathione, 0.25 mM DTT, 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 PKA C alpha (0.1 μg/μL) diluted with Kinase Dilution Buffer VII to the concentrations indicated in Figure 2. 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 VII - Kinase Assay Buffer I diluted at a 1:4 ration (5X dilution) with 50 ng/μL BSA and 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. 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. Store 1 mL aliquots at ≤ -20 °C.
  • Substrate - CREBtide synthetic peptide substrate (KRREILSRRPSYR) diluted in distilled 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 Active PKA C alpha , Kinase Assay Buffer, 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 Active PKA C alpha : 10 μL
     b. Stock solution of Substrate (1 mg/mL): 5 μL
     c. Distilled water (2-8 °C): 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 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)]

Notes

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

Alternate Names for Recombinant Human Active PKA C alpha Protein, CF

• cAMP-dependent protein kinase catalytic subunit alpha
• Cs
• EC 2.7.11
• EC 2.7.11.11
• MGC102831
• MGC48865
• PKA C alpha
• PKA C-alpha
• Pkaca
• PKCA1
• PKCD
• PRKACA
• protein kinase A catalytic subunit
• protein kinase, cAMP-dependent, catalytic, alpha

Background

The catalytic subunit C-alpha of PKA (PKA C alpha ) is a member of the Ser/Thr protein kinase family and has been assigned to chromosome region 19p13.1 (1). Null mutation in PKA C alpha leads to early postnatal lethality in the majority of C-alpha knockout mice. Surprisingly, a small percentage of C-alpha knockout mice, although runted, survived to adulthood. In these animals, compensatory increases in C-beta levels occurred in brain whereas many tissues, including skeletal muscle, heart, and sperm contained less than 10% of the normal PKA activity (2).

1. Tasken, K. et al. (1996) Genomics 36:535.
2. Skalhegg, B.S. et al. (2002) Molec. Endocr. 16:630.

Publications for PKA C alpha (4268-KS)(1)

Publication using 4268-KS

• The GCN5-CITED2-PKA signalling module controls hepatic glucose metabolism through a cAMP-induced substrate switch Nat Commun, 2016-11-22;7(0):13147. 2016-11-22 [PMID: 27874008] (Human)

Bioinformatics

• Gene Symbol: PRKACA
• Uniprot:
  • NM_002730(Human)