tubulin Protocols

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CHO Centrosome Prep Protocol - http://mitchison.med.harvard.edu/protocols/mt1.html

Category: Cell Organelle Protocols: Nucleus Protocols

The key step is the lysis which solubilizes centrosomes away from nuclei by very low ionic strength lysis after treatment of cells with nocodazole and cytochalasin B. The released centrosomes are then centrifuged onto a Ficoll cushion (to avoid pelleting) and the interface between the lysate and the Ficoll is collected and the centrosomes are concentrated on a sucrose gradient. Fractions are assayed by spindown and double IF with 5051 serum and anti-tubulin and the pooled fractions are frozen... - [Read CHO Centrosome Prep Protocol]

Confocal Microscopy Specimen Preparation Using Synthetic Fluorophores and Immunofluorescence - http://www.olympusconfocal.com/applications/protocols/index.html

Category: Microscopy Protocols: Confocal Microscopy Protocols

Includes staining protocols: Triple-Staining Adherent Cells with MitoTracker, Phalloidin,& Nuclear Dyes; Staining Adherent Cells with Cytokeratin Primary Antibodies & Synthetic Fluorophores; Staining Adherent Cells with Intermediate Filament Primary Antibodies & Synthetic Fluorophores; Staining Cells & Tissue Cryosections with Tubulin Primary Antibodies, Phallotoxins,& Synthetic Fluorophores; Triple-Staining Tissue Cryosections with Wheat Germ Agglutinin, Phalloidin,& Nuclear Dyes; etc. - [Read Confocal Microscopy Specimen Preparation Using Synthetic Fluorophores and Immunofluorescence]

Immunofluorescent Localization of Tubulin Protocol - http://homepages.gac.edu/~cellab/chpts/chpt2/ex2-7.html

Category: Cytoskeleton Protocols: Microtubules Protocols

Protocol for the immunoflourescent localization of tubulin. - [Read Immunofluorescent Localization of Tubulin Protocol]

Microtubule Assembly Protocol - http://www.proweb.org/kinesin/Methods/MT_assembly.html

Category: Cell Biology and Cell Culture

Protocol on microtubule assembly using purified tubulin. - [Read Microtubule Assembly Protocol]

Microtubule/ organelle Motility Assays - http://www.bio.unc.edu/faculty/salmon/lab/protocolsmtorganellemotilityassays.html

Category: Cell Biology and Cell Culture

Microtuble/ organelled motility assay using Golgi or ER membranes, 45 uM tubulin, rat liver cytosol, and 20x energy regeneration system. - [Read Microtubule/ organelle Motility Assays]

Porcine Brain Tubulin Prep Protocol - http://www.bio.unc.edu/faculty/salmon/lab/protocolsporcinetubulin.html

Category: Cytoskeleton Protocols: Microtubules Protocols

The protocol calls for 3 pig brains, and should yield ~ 60 mg of purified tubulin. - [Read Porcine Brain Tubulin Prep Protocol]

Protocol for Cycling Tubulin - http://www.ciwemb.edu/labs/koshland/Protocols/MICROTUBULE/cyclingmicrotub.html

Category: Cell Biology and Cell Culture

Protocol for cycling tubulin using the buffers Glycerol PB and BRB80. - [Read Protocol for Cycling Tubulin]

Protocol for Microtubule Assembly - http://www.bio.com/protocolstools/protocol.jhtml?id=p1600

Category: Cell Biology and Cell Culture

Purified Tubulin is polymerized and then stabilized with Taxol. The resulting Microtubules are then ready for use in motility assays. Protocol use solutions such as 50 mM Mg-GTP, 50 Î¼l 100 mM GTP, 40 Î¼M Taxol, Taxol Stock,and PM Buffer. - [Read Protocol for Microtubule Assembly]

Protocol for Motor Polarity Using Fluorescent Microtubules - http://www.proweb.org/kinesin/Methods/Fluorescent_MT.html

Category: Cell Biology and Cell Culture

Protocol for motor polarity using fluorescent microtubules using the materials: Rhodamine-tubulin (Molecular Probes),NEM- tubulin, PC-tubulin, MgÂ·GTP, MgCl2 and Glycerol. - [Read Protocol for Motor Polarity Using Fluorescent Microtubules]

Protocol Spindle Assembly In Vitro - http://www.bio.com/protocolstools/protocol.jhtml?id=p1051

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes an in vitro reaction to assay mitotic spindle assembly. The assay uses Cytostatic Factor extract made from Xenopus eggs, fluorescently-labelled tubulin, and prepared sperm nuclei. Spindle assembly is monitored by immunofluorescence microscopy. - [Read Protocol Spindle Assembly In Vitro]

Recycling Tubulin Protocol - http://mitchison.med.harvard.edu/protocols/recycle.html

Category: Cytoskeleton Protocols: Microtubules Protocols

Recycle tubulin fractions stored at -80¡C after the PC column and store the recycled tubulin in small aliquots for day-to-day use. Generally store recycled tubulin in Injection Buffer (IB) without free GTP. This is done because depolymerization appears to be much better in IB, IB is ideal for microinjections/adding tubulin to extracts, and the absence of free GTP makes polymerization with GMPCPP, a very useful GTP analog that has ~5-10X lower affinity than GTP for tubulin. - [Read Recycling Tubulin Protocol]

Tubulin Polymerization with GTP/GMPCPP/Taxol Protocol - http://mitchison.med.harvard.edu/protocols/poly.html

Category: Cytoskeleton Protocols: Microtubules Protocols

Protocol for tubulin polymerization with GTP/GMPCPP/ Taxol. Includes: Solutions & Supplies; Prepolymerization Clarification; GTP Polymerization; Taxol Polymerization; GMPCPP Polymerization; Determining Concentration of GMPCPP/Taxol MTs. - [Read Tubulin Polymerization with GTP/GMPCPP/Taxol Protocol]

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Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.