Measuring Protein Binding to F-actin by Co-sedimentation

作者：Heier Jonathon A; Dickinson Daniel J; Kwiatkowski Adam V^*

来源：Jove-Journal of Visualized Experiments, 2017, (123): e55613.

DOI：10.3791/55613

• Biochemistry
• Issue 123
• actin
• filamentous actin
• F-actin
• actin-binding protein
• co-sedimentation
• pelleting assay
• binding curve

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique - the actin co-sedimentation, or pelleting, assay - to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

• 出版日期2017-5