Chapter title |
Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries
|
---|---|
Chapter number | 15 |
Book title |
Kinase Screening and Profiling
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3073-9_15 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3072-2, 978-1-4939-3073-9
|
Authors |
Chad J. Miller, Benjamin E. Turk |
Abstract |
Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 25 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Doctoral Student | 3 | 12% |
Researcher | 3 | 12% |
Student > Bachelor | 2 | 8% |
Student > Ph. D. Student | 2 | 8% |
Student > Master | 2 | 8% |
Other | 3 | 12% |
Unknown | 10 | 40% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 6 | 24% |
Agricultural and Biological Sciences | 3 | 12% |
Unspecified | 1 | 4% |
Pharmacology, Toxicology and Pharmaceutical Science | 1 | 4% |
Social Sciences | 1 | 4% |
Other | 2 | 8% |
Unknown | 11 | 44% |