Development and Validation of a Competitive AKT Serine/Threonine Kinase Fluorescence Polarization Assay Using a Product-Specific Anti-phospho-serine Antibody

2001 ◽  
Vol 299 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Tammy C. Turek ◽  
Eliza C. Small ◽  
Robert W. Bryant ◽  
W. Adam ◽  
G. Hill
Keyword(s):  
Fluorescence Polarization ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Fluorescence Polarization Assay ◽  
Development And Validation

scholarly journals Development of a Microplate-Based, Electrophoretic Fluorescent Protein Kinase A Assay: Comparison with Filter-Binding and Fluorescence Polarization Assay Formats

2005 ◽  
Vol 10 (4) ◽  
pp. 329-338 ◽  
Author(s):  
Siobhan M. Miick ◽  
Shila Jalali ◽  
Brian P. Dwyer ◽  
John Havens ◽  
Donald Thomas ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Fluorescence Polarization ◽  
Fluorescent Protein ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Kinase Reaction ◽  
Fluorescence Polarization Assay ◽  
Phosphorylated Peptide ◽  
Kinase A

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture™ PKA assay developed uses a positively charged, lissamine-rhodamine–labeled kemptide peptide substrate for the kinase reaction and Nanogen’s ElectroCapture™ HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture™ PKA assay was validated with both known PKA inhibitors and library compounds. The pKiapp results obtained in the ElectroCapture™ PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.

Development of a Transcreener™ Kinase Assay for Protein Kinase A and Demonstration of Concordance of Data with a Filter-Binding Assay Format

2007 ◽  
Vol 12 (4) ◽  
pp. 578-584 ◽  
Author(s):  
Karen L. Huss ◽  
Pauline E. Blonigen ◽  
Robert M. Campbell
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Fluorescence Polarization ◽  
Binding Assay ◽  
Kinase Assay ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Alexa Fluor ◽  
Small Molecule Library ◽  
Kinase A

A Transcreener™ kinase fluorescence polarization (FP) assay has been developed for the serine/threonine kinase protein kinase A (PKA). The PKA Transcreener™ kinase assay is an homogenous, competitive antibody-based FP assay that uses Far Red Alexa Fluor 633-labeled adenosine 5′ disphosphate (ADP) tracer and mouse monoclonal anti-ADP antibody. The Transcreener™ PKA assay was validated with both known PKA inhibitors and library compounds. The Transcreener™ PKA assay is resistant to low-wavelength (or common) fluorescent interference from small-molecule library compounds and generates IC50 results comparable with current radioactive filter-binding assay. ( Journal of Biomolecular Screening 2007:578-584)

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

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