Product Pathways - HTScan Kinase Assay Kits
HTScan® SGK3 Kinase Assay Kit #7621
Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.
| Kit Includes | Quantity |
|---|---|
| PLK (Ser137) Biotinylated Peptide # 1300 | 1.25 milliliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| Phospho-PLK (Ser137) Antibody # 5070 | 30 microliters |
| SGK3 Kinase # 7620 | 5 micrograms |
| ATP (10 mM) # 9804 | 1 milliliters |
Description
The kit provides a means of performing kinase activity assays with recombinant full-length human SGK3 kinase. It includes active SGK3 kinase (supplied as a GST fusion protein), a biotinylated peptide substrate and a phospho-serine/threonine antibody for detection of the phosphorylated form of the substrate peptide.
Molecular Weights
Peptide substrate, Biotin-PLK (Ser137): 1,945 Daltons. GST-SGK3 Kinase: 83 kDa.
Peptide Core Sequence
RRS*LL
Kinase Assay - Radiometric
Figure 1. SGK3 kinase activity was measured in a radioisotopic filter binding assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 1 µM ATP, 2.5 µg/50 µl PEG20,000, Substrate: GST-GSK3 (14-27), variable and recombinant SGK3, variable.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, increasing amounts of SGK3 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 5. Staurosporine inhibition of SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of SGK3 substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, 50 ng SGK3, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 4. Peptide concentration dependence of SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, 50 ng of SGK3 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Kinase Assay - DELFIA
Figure 2. Time course of SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of SGK3 substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, 50 ng SGK3 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full-length human SGK3 (Met1-Leu496) (GenBank Accession No. NM_013257.3) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Quality Control
The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-PLK (Ser137) Antibody #5070 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified SGK3 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain and Western blot. The specific activity of the SGK3 kinase was determined using a radiometric filter binding assay [Fig.1]. Time course [Fig.2], kinase dose dependency [Fig.3] and substrate dose dependency [Fig.4] assays were performed to verify SGK3 activity using the SGK3 substrate peptide provided in this kit. SGK3 sensitivity to the inhibitor staurosporine was measured using the SGK3 substrate peptide provided in this kit [Fig.5].
Background
Serum and glucocorticoid-inducible kinase (SGK) is a serine/threonine kinase closely related to Akt (1). SGK is rapidly induced in response to a variety of stimuli, including serum, glucocorticoid, follicle stimulating hormone, osmotic shock and mineralocorticoids. SGK activation can be accomplished via HGF PI3K-dependent pathways and by integrin-mediated PI3K-independent pathways (2,3). Induction and activation of SGK has been implicated in activating the modulation of antiapoptotic and cell cycle regulation (4-6). SGK also plays an important role in activating certain potassium, sodium and chloride channels, suggesting its involvement in the regulation of processes such as cell survival, neuronal excitability and renal sodium excretion (2). SGK is negatively regulated by ubiquitin modification and proteasome degradation (7).
- Webster, M.K. et al. (1993) Mol. Cell. Biol. 13, 2031-2040.
- Kobayashi, T. and Cohen, P. (1999) Biochem. J. 339, 319-328.
- Park, J. et al. (1999) EMBO J. 18, 3024-3033.
- Brunet, A. et al. (2001) Mol. Cell. Biol. 21, 952-965.
- Mikosz, C.A. et al. (2001) J. Biol. Chem. 276, 16649-16654.
- Hayashi, M. et al. (2001) J. Biol. Chem. 276, 8631-8634.
- Brickley, D.R. et al. (2002) J. Biol. Chem. 277, 43064-43070.
Application References
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
Companion Products
- 5070 Phospho-PLK (Ser137) Antibody
- 1300 PLK (Ser137) Biotinylated Peptide
- 7620 SGK3 Kinase
- 9802 Kinase Buffer (10X)
- 9804 ATP (10 mM)
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
