Gel-Based Methods for the Investigation of Signal Transduction Pathways in Trypanosoma brucei

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2116; pp. 497 - 522
• Main Authors: Szöőr, Balázs, Cayla, Mathieu
• Format: Book Chapter Journal Article
• Language: English
• Published: New York, NY Springer US 2020
• Series: Methods in Molecular Biology
• Subjects: Chelating Agents - chemistry; Electrophoresis, Polyacrylamide Gel - methods; Kinase assays; Phos-tag; Phospho substrate; Phosphoproteins - isolation & purification; Phosphoproteins - metabolism; Phosphorylation; Protein kinases; Protein Kinases - metabolism; Protein phosphatases; Protozoan Proteins - isolation & purification; Protozoan Proteins - metabolism; Pyridines - chemistry; SDS-PAGE; Signal Transduction; Staining and Labeling - methods; Trypanosoma brucei brucei - metabolism; SDS-PAGE; Signal transduction; Kinase assays; Phos-tag; Protein phosphatases; Phospho substrate; Protein kinases
• ISBN: 1071602934; 9781071602935
• ISSN: 1064-3745; 1940-6029
• DOI: 10.1007/978-1-0716-0294-2_30

In the cell, reversible phosphorylation, controlled by protein phosphatases and protein kinases, initiates and regulates various signaling-dependent processes such as enzyme–substrate interactions, the cell cycle, differentiation, and immune responses. In addition to these processes, in unicellular parasites like Trypanosoma brucei, the causative agent of African sleeping sickness, additional signaling pathways have evolved to enable the survival of parasites in the changing environment of the vector and mammalian host. In this chapter, we describe two in vitro kinase assays and the use of the phosphoprotein chelator Phos-tag and show that these three polyacrylamide gel-based assays can be used for rapid target validation and detection of changes in phosphorylation.