Product Pathways - Akt Signaling
HTScan® PDK1 Kinase Assay Kit #7577
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 |
|---|---|
| PDK1 Kinase # 7386 | 5 micrograms |
| PKA (Thr197) Biotinylated Peptide # 1024 | 1.25 milliliters |
| Kinase Buffer (10X) # 9802 | 15 milliliters |
| ATP (10 mM) # 9804 | 1 milliliters |
| Phospho-PKA C (Thr197) Antibody # 4781 | 30 microliters |
Description
The kit provides a means of performing kinase activity assays with recombinant human PDK1 kinase. It includes active PDK1 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
Biotin-peptide: 2,205 Daltons. PDK1 Kinase: 67 kDa.
Peptide Core Sequence
RTWT*LCG
Kinase Assay - Radiometric
Figure 1. PDK1 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 ng/μl BSA, 50 μM ATP, Substrate: PDKtide 400 ng/μL, and recombinant PDK1: variable.
Kinase Assay - DELFIA
Figure 3. Dose dependence curve of PDK1 kinase activity: DELFIA® data generated using Phospho-PKA C (Thr197) Antibody #4781 to detect phosphorylation of substrate peptide (#1024) by PDK1 kinase. In a 50 µl reaction, increasing amounts of PDK1 and 3.0 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 PDK1 kinase activity: DELFIA® data generated using Phospho-PKA C (Thr197) Antibody #4781 to detect phosphorylation of PDK1 substrate peptide (#1024) by PDK1 kinase. In a 50 µl reaction, 50 ng PDK1, 3.0 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 PDK1 kinase activity: DELFIA® data generated using Phospho-PKA C (Thr197) Antibody #4781 to detect phosphorylation of substrate peptide (#1024) by PDK1 kinase. In a 50 µl reaction, 50 ng of PDK1 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 PDK1 kinase activity: DELFIA® data generated using Phospho-PKA C (Thr197) Antibody #4781 to detect phosphorylation of PDK1 substrate peptide (#1024) by PDK1 kinase. In a 50 µl reaction, 50 ng PDK1 and 3.0 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Source / Purification
The Kinase fusion protein was produced using a baculovirus expression system using sf9 cells and a recombinant virus encoding for the full length human PDK1 (Met1-Met460) (GenBank Accession No. NM_002613) with an amino-terminal His tag. The protein was purified by Immobilized Metal Affinity Chromatography (IMAC).
Quality Control
The substrate peptide was selected using our Serine/Threonine Kinase Substrate Screening Kit #7400. Phospho-PKA C (Thr197) Antibody #4781 was used for detection. The quality of the biotinylated peptide was evaluated by reverse-phase HPLC and by mass spectrometry.Purified PDK1 kinase was quality controlled for purity by SDS-PAGE followed by Coomassie stain. PDK1 kinase activities were determined using a radiometric assay [Fig.1]. Time course [Fig.2], kinase dose-dependency [Fig.3] and substrate dose-dependency [Fig.4] assays were performed to verify PDK1 activity using the PDK1 substrate peptide provided in this kit. PDK1 sensitivity to the inhibitor staurosporine was measured using the PDK1 substrate peptide provided in this kit [Fig.5].
Background
Phosphoinositide-dependent protein kinase 1 (PDK1) plays a central role in many signal transduction pathways (1,2), activating Akt and the PKC isoenzymes p70 S6 kinase and RSK (3). Through its effects on these kinases, PDK1 is involved in the regulation of a wide variety of processes, including cell proliferation, differentiation and apoptosis.
- Belham, C. et al. (1999) Curr. Biol. 9, R93-R96.
- Toker, A. and Newton, A.C. (2000) Cell 103, 185-188.
- Williams, M.R. et al. (2000) Curr. Biol. 10, 439-448.
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
- 7386 PDK1 Kinase
- 4781 Phospho-PKA C (Thr197) Antibody
- 1024 PKA (Thr197) Biotinylated Peptide
- 9953 Staurosporine
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.
