Computational protocol: Motile properties of the bi-directional kinesin-5 Cin8 are affected by phosphorylation in its motor domain

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Protocol publication

[…] In vitro motility assays were performed following procedures we previously described in MB of the desired salt concentration supplemented with 10 μM Taxol, 0.1 mg/ml glucose oxidase, 0.8 mg/ml catalase, 1 mM DTT, 10 mM glucose, 0.2 mg/ml casein, 4 mM MgCl2, and 4 mM ATP or ADP. MTs were polymerized with TMR-labelled tubulin (TL590M Cytoskeleton Inc.), and polarity marked using Atto-488-labelled seeds or seeds with a significantly higher concentration of TMR-labelled tubulin marking the MT minus-end. Single molecule fluorescence data were collected on a Zeiss Axiovert 200 M-based microscope setup equipped with an HBO 100 Mercury Illuminator, and a cooled CCD camera (SensiCam, PCO or Andor Neo sCMOS) with frame time of 1 s. Data was processed using ImageJ and MetaMorph (MDS Analytical Technologies) software. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Saccharomyces cerevisiae