Fluorescent peptide assays for protein kinases.
Protein kinases are enzymes that regulate many cellular events in eukaryotic cells, such as cell-cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP-binding site, as well as one or more substrate-binding site(s) that exhibit recognition features for a protein substrate. Thus, by bringing ATP and a substrate into close proximity, each protein kinase can modify its substrate by transferring the gamma phosphate of the ATP molecule to a serine, threonine, or tyrosine residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated, dependent on the phosphorylated versus dephosphorylated forms of the substrate. This unit describes an assay employing a fluorescent peptide substrate to measure the incorporation of non-radiolabeled phosphate. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore.
Devkota
Ashwini K
AK
University of Texas at Austin, Austin, Texas, USA.
Kaoud
Tamer S
TS
Warthaka
Mangalika
M
Dalby
Kevin N
KN
D005453:Fluorescence; D005456:Fluorescent Dyes / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism*; D010758:Phosphorus / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D010455:Peptides; D010758:Phosphorus; D011494:Protein Kinases