Protocols and Software
ASSAYS TO STUDY MICROTUBULE MOTIONS IN VITRO:
- Manufacturing reusable flow chambers
- Preparation of coverslips
- Preparation of GMPCPP-stabilized microtubule seeds
- Attachment of microtubule seeds to the coverslips
- Preparation of segmented microtubules.
Video illustrating protocols 1-5 (Volkov et al., 2014)
- Experimental observation of the protein tracking with depolymerizing microtubule ends
- Preparation of streptavidin-coated beads
- Preparation of unevenly coated beads
- Recording the photobleaching kinetics for the fluorescently labeled protein molecules
APPROACHES TO RECONSTRUCT AND ANALYZE KINETOCHORES IN VITRO
Protein purifications
Microscopy assays
- Quantitative analysis of fluorescence intensities of mi3-based particles
- Quantification of the photobleaching rate for mi3-based particles with dual-color proteins
- Binding of fluorescent proteins to mi3-based particles with dual color proteins
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TIRF microscopy assay to study the interactions between monomeric CENP-T and soluble Ndc80
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TIRF microscopy assay to study the interactions between clustered CENP-T and soluble Ndc80
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Quantitative analysis of the molecular size of fluorescent molecules and clusters
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Analysis of photobleaching rate of monomeric and clustered fluorescent molecules
ASSAYS TO STUDY DENSE GRANULE SECRETION AND CALCIUM OSCILLATIONS IN INDIVIDUAL PLATELETS
Protocols
- Fluorescent measurement of dense granules and intracellular calcium in individual platelets in microfluidic flow chambers
- Immobilization of PECAM-1 antibody on the coverslips of the microfluidic chambers
- Quantitative analysis of dense granules and intracellular calcium in individual platelets
Source codes
- Quantitative analysis of kinetics of dense granule secretion and calcium oscillations in individual platelets (Manual | Source Code)
- Quantitative analysis of kinetics of dense granule secretion and calcium oscillations in platelet population (Source Code)
IMAGE ANALYSIS
- Correcting for unevenness of illumination
- Determining the intensity of single fluorophore
- Quantification of bead brightness Protocol | Source Code
SOFTWARE
- CENP-E tip tracking (Gudimchuk et al., 2012) Manual | Source Code
- Molecular-mechanical model of the dynamic microtubule (Zakharov et al., 2015) Manual | Source Code
- Tethered Cargo Motion (TCM) Monte Carlo simulation software (Gudimchuk et al., 2018) Manual | Source Code