For cell cycle experiments, cells were arrested by adding 10 μM of the CDK1 inhibitor RO3306 (Merck) for 18 h before washed out and release (2 h) to conduct experiments when early G1 was reached. Subcellular fractionations and phalloidin‐based pulldowns were performed as described previously (Baarlink et al, 2017 ). Briefly, cells were pelleted and resuspended before vortexing and centrifugation. The supernatant was used to obtain the cytoplasmic extract. The pelleted nuclei were extracted in lysis buffer before sonication and centrifugation to obtain the nucleoplasmic fraction. Immunoblotting was performed with α‐tubulin as cytoplasmic and histone H3 (Cell Signaling) as nucleoplasmic marker to verify successful fractionation. For co‐immunoprecipitations, agarose beads conjugated with Flag‐M2 antibody (Sigma‐Aldrich) were equilibrated in lysis buffer before subcellular fractionations were loaded onto the beads at 4°C for 90 min. Beads were resuspended processed for Western analysis. Phalloidin pulldown assays were performed with streptavidin magnetic Dynabeads (Thermo Fisher Scientific) and Biotin‐Phalloidin (Thermo Fisher Scientific).
Biotin phalloidin
Manufactured by Thermo Fisher Scientific
Biotin-Phalloidin is a fluorescent conjugate used to label and detect filamentous actin (F-actin) in biological samples. It binds specifically to F-actin and can be visualized using fluorescence microscopy.
Automatically generated - may contain errors
3 protocols using biotin phalloidin
1
Cell Cycle-Synchronized Subcellular Fractionation
2
Quantifying Utrophin-Actin Binding Kinetics
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To evaluate the binding properties of different utrophin ABD mutants, single molecule binding kinetics were measured. Actin filaments were polymerized at a final concentration of 5 µM at room temperature in assay buffer (25 mM Immidizole, 25 mM KCl, 4 mM MgCl2, 1 mM EGTA, 1 mM DTT, pH 7.4). To immobilize actin filaments to the surface of the flow chamber, flow wells were first incubated with 10 µg/mL streptavidin (Sigma #S0677) for 1 min, washed with assay buffer and then incubated with 1 µM biotin phalloidin, (ThermoFisher #B7474) for 1 min. Actin filaments were then diluted 50× in assay buffer and immediately introduced into the flow well and allowed to attach for 5 min. To prevent shearing of filaments, pipette tips were cut when pipetting actin filaments. Remaining filaments were washed away with assay buffer containing Beta Casein (25 mM Immidizole, 25 mM KCl, 4 mM MgCl2, 1 mM EGTA, 1 mM DTT and 10 µg/mL Beta Casein (Sigma C6905)). Binding proteins were diluted to a sufficiently low concentration to enable the visualisation of single molecules in TIRF, 0.05–10 nM in assay buffer. For single molecule kinetic measurements 600 frames were acquired at an interval of 30–130 ms depending on the construct.
3
Cell Cycle Synchronization and Fractionation
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For cell cycle experiments, cells were arrested by adding 10 µM of the CDK1 inhibitor RO3306 (Merck) for 18 h before washed out and release (2h) to conduct experiments when early G1 was reached. Subcellular fractionations and phalloidin-based pull-downs were performed as described previously (Baarlink et al., 2017) . Briefly, cells were pelleted and resuspended before vortexing and centrifugation. The supernatant was used to obtain the cytoplasmic extract. The pelleted nuclei were extracted in lysis buffer before sonication and centrifugation to obtain the nucleoplasmic fraction. Immunoblotting was performed with α-tubulin as cytoplasmic and histone H3 (Cell Signaling) as nucleoplasmic marker to verify successful fractionation. For coimmunoprecipitations, agarose beads conjugated with Flag-M2 antibody (Sigma-Aldrich) were equilibrated in lysis buffer before subcellular fractionations were loaded onto the beads at 4°C for 90 min. Beads were resuspended processed for Western analysis. Phalloidin pulldown assays were performed with streptavidin magnetic Dynabeads (Thermo Fisher Scientific) and Biotin-Phalloidin (Thermo Fisher Scientific).
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