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

Catalog # Availability Size / Price Qty
3200-KS-010
Recombinant Human Active SGK1 (60-end) Protein, CF SDS-PAGE
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Recombinant Human Active SGK1 (60-end) Protein, CF Summary

Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Activity
The activity of SGK1 is 99-135 nmol/min/mg using a synthetic peptide substrate (RPRAATF) (see Activity Assay Protocol).
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human SGK1 protein
aa 60-431
Accession # NM_005627
With an N-terminal GST tag
Accession #
SDS-PAGE
73 kDa

Product Datasheets

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

3200-KS

Formulation 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.
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

Materials
  • Active Kinase - Active SGK1 (0.1 μg/μL) diluted with Kinase Dilution Buffer and assayed as outlined in Figure 2. 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 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.
  • [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.
  • Substrate - SGK synthetic peptide substrate (RPRAATF) 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
  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 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 phosphocellulose P81 paper.
  7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10 mL of phosphoric acid and make a 1 liter solution with 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 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)]

Data Image

SDS-PAGE View Larger

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

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Background: SGK1

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.

References
  1. Kobayashii, T. et al. (1999) Biochem. J. 1:189.
  2. Brunet, A. et al. (2001) Mol. Cell. Biol. 21:952.
Long Name
Serum/Glucocorticoid Regulated Kinase 1
Entrez Gene IDs
6446 (Human); 20393 (Mouse); 29517 (Rat)
Alternate Names
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

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