Negative Stain Electron Microscopy of Microtubules Protocol Negative staining is a rapid, qualitative method for analyzing microtubule structure at the EM level. Because negative staining involves deposition of heavy atom stains, structural artifacts such as flattening of the cylindrical microtubule and opening up of microtubules into flat sheets are common. Negative staining is very useful because of its ease, rapidity and lack of requirement for specialized equipment other than that found in a regular EM facility.
Preparation of Segmented and Polarity Marked Microtubules Protocol Segmented and polarity-marked microtubules are very useful for many different types of in vitro assays. Segmented microtubules are microtubules with a bright seed and dim elongated segments on both ends. Polarity marked microtubules are microtubules with a bright seed and a dim elongated segment only on one end -- the plus end.
Recycling Tubulin Protocol 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.
Tubulin Polymerization with GTP/GMPCPP/Taxol Protocol 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.
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