Recombinant Human Active SGK1 (60-end) Protein, CF

The approximate molecular weight is 73 kDa and the average purity is 95%.

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

Recombinant Human Active SGK1 (60-end) Protein, CF Summary

• Details of Functionality: The specific activity of SGK1 was determined to be 99 nmol/min/mg using a Akt (PKB) peptide substrate as per activity assay protocol.
• Source: Spodoptera frugiperda, Sf 9 (baculovirus)-derived human SGK1 protein
  aa 60-431
  With an N-terminal GST tag
• Accession #: NM_005627
• Protein/Peptide Type: Recombinant Proteins
• Gene: SGK1
• 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: 73 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, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 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 SGK1 (0.1 μg/μL) diluted with Kinase Dilution Buffer 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 -glycerol-phosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer I prior to use.
  • Kinase Dilution Buffer - Kinase Assay Buffer I diluted at a 1:4 ratio (5-fold dilution) with distilled or deionized water.
  • 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 1.0 mL aliquots at ≤ -20°C.
  • Substrate - Akt (PKB) peptide substrate (CKRPRAASFAE) 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 SGK1, 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 Active SGK1: 10 μL
     b. Substrate (1 mg/mL Stock Solution): 5 μL
     c. Distilled water: 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 water.
  5. Initiate the reaction by 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 period, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phospho cellulose 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 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 removing 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 SGK1 (60-end) Protein, CF

• EC 2.7.11
• EC 2.7.11.1
• serine/threonine protein kinase SGK
• serine/threonine-protein kinase Sgk1
• serum/glucocorticoid regulated kinase 1
• serum/glucocorticoid regulated kinase
• Serum/glucocorticoid-regulated kinase 1
• SGK
• SGK1

Background

SGK1 is a member of the serum- and glucocorticoid-induced protein kinase that is activated in vitro by 3-phosphoinositide-dependent protein kinase-1 (PDK 1) and in vivo in response to signals that activate phosphatidylinositol (PI) 3-kinase (1). SGK1 mRNA is expressed in all tissues and the level of SGK1 mRNA is increased by stimulation with serum or dexamethasone. SGK1 promotes cell survival by phosphorylating and inactivating FKHRL1 (2). SGK and Akt display differences with respect to the efficacy with which they phosphorylate the three regulatory sites on FKHRL1.

1. Kobayashii, T. et al. (1999) Biochem. J. 1:189.
2. Brunet, A. et al. (2001) Mol. Cell. Biol. 21:952.

Bioinformatics

• Gene Symbol: SGK1
• Uniprot:
  • NM_005627(Human)