In Vitro Methods to Study AMPK.
AMPK studies in cell-free and cellular systems have significantly contributed to recent progress in the AMPK field. Biochemical characterization, structure determination and elucidation of AMPK-dependent signalling events benefit from application of state-of-the-art tools and methodology. This chapter provides a synopsis of recombinant protein expression systems and biochemical and cell-based study methods. We summarize three different expression systems for AMPK production: bacteria, insect cells and mammalian cells. In addition, kinase activity measurement, kinase substrate identification and determination of physical interaction with AMPK are discussed. The last part of this chapter focuses on the use of pharmacological activation, inhibition, and molecular genetic tools to study AMPK involvement in cellular signalling pathways.
Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University Medical Centre, 616, 6200 MD, Maastricht, The Netherlands.
Xiaoqing
X
Zhu
Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University Medical Centre, 616, 6200 MD, Maastricht, The Netherlands.
J Willem
JW
Voncken
Department of Molecular Genetics, CARIM School for Cardiovascular Diseases, Maastricht University Medical Centre, 616, 6200 MD, Maastricht, The Netherlands. d.neumann@maastrichtuniversity.nl.
Dietbert
D
Neumann
D055372:AMP-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System / Q000378:metabolism; D057075:Enzyme Assays; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D015398:Signal Transduction / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D021122:Protein Subunits; D011994:Recombinant Proteins; D055372:AMP-Activated Protein Kinases
AMPK activity modulation; Genetic tools; Kinase activity determination; Recombinant protein expression; Substrate identification