UID:
almafu_9958126775602883
Format:
1 online resource (539 p.)
Edition:
First edition.
ISBN:
0-12-398480-7
Series Statement:
Methods in Enzymology, Volume 540
Content:
This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers cytoskeletal structure, including such topics as rotational movement of formins studied by fluorescence polarization microscopy, in vitro assembly assays for bacterial actin filaments, and modulators of microtubule plus end growth.Continues the legacy of this premier serial with quality chapters authored by leaders in the fieldCovers cytoskeletal structureContains chapters with such topics as rec
Note:
Description based upon print version of record.
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Front Cover; Reconstituting the Cytoskeleton; Copyright; Contents; Contributors; Preface: The Role of Reconstitution in Cytoskeleton Research; Acknowledgment; References; Section I: Polymer Dynamics; Chapter One: Actin Filament Dynamics Using Microfluidics; 1. Introduction; 2. Key Technical Aspects of the Microfluidic Method; 2.1. Overview; 2.2. Controlling the flow; 2.3. Controlling surface properties; 2.4. Calibrating the applied force; 2.5. Caveats and control experiments; 3. Assets for the Study of Actin Dynamics; 3.1. Improving the observation of single filaments
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3.2. New experimental capabilities3.2.1. Kinetic measurements and more; 3.2.2. Building hybrid filaments; 3.2.3. Observing subpopulations of filaments; 3.2.4. Applying mechanical constraints to many filaments in parallel; 4. Perspectives; 4.1. Expanding the setup; 4.1.1. Orienting filaments toward micropatterns or structures; 4.1.2. Lipid layers; 4.1.3. Coupling to other techniques; 4.2. Beyond single-filament actin dynamics; 4.2.1. Other cytoskeletal filaments; 4.2.2. Motor proteins; 4.2.3. Filament networks; References
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Chapter Two: Bacterial Actin-Like Proteins: Purification and Characterization of Self-Assembly Properties1. Introduction; 1.1. Protein expression and purification; 1.1.1. Expression; 1.1.2. Protein purification; 1.1.3. Buffers; 1.2. Bulk measurements; 1.2.1. Light scattering versus FRET; 1.2.2. Light scattering; 1.2.3. FRET; 1.2.3.1. Critical concentration versus steady-state monomer concentration; 1.2.3.2. Nucleation; 1.2.3.3. Dynamic instability; 1.2.4. Negative stain electron microscopy; 1.3. TIRF: Visualizing filament dynamics; 1.3.1. Labeling strategies
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1.3.2. Surface chemistry: APTES-PEG-biotin glass1.3.2.1. Silanized glass; 1.3.2.2. Pegylation; 1.3.3. TIRF microscopy; 1.3.4. Polarity; 1.3.4.1. Making cross-linked stable seeds; 1.4. Other considerations; 1.4.1. Nucleotide affinity and preference; 2. Conclusion; Acknowledgments; References; Chapter Three: Quantitative Analysis of Microtubule Self-assembly Kinetics and Tip Structure; 1. Introduction; 2. Tip-Tracking Algorithm Walkthrough; 2.1. Fitting the microtubule backbone; 2.2. Measuring tip position; 2.3. Measuring tip structure; 3. In Vivo Tip-Tracking Protocol
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3.1. Cell culture and imaging3.2. Experimental setup considerations; 3.2.1. Signal above background; 3.2.2. Spatial sampling; 4. TipTracker Optimization Using Fixed Cells; 5. Considerations for In Vitro Assays and Other Filament Systems; Acknowledgments; References; Section II: Polymer Nucleation and Regulation; Chapter Four: Biochemical Reconstitution of the WAVE Regulatory Complex; 1. Introduction; 2. Overview of the In Vitro Reconstitution Method; 3. Key Reagents; 4. Reconstitution of the WAVE/Abi/HSPC300 Trimer; 4.1. Expression and purification of individual trimer components
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4.1.1. Expression constructs for trimer proteins
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English
Additional Edition:
ISBN 0-12-397924-2
Language:
English
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