Chapter 26 High-Resolution Negative Staining of the Isolated Dynein ATPase

Abstract

Publisher Summary Negative staining discussed in this chapter is meant for studying the ultra structure of intact axonemes and isolated axonemal proteins. It has the advantage that the whole technique from initial preparation to final examination takes only minutes and does not require the use of any exotic equipment, other than a transmission electron microscope and a carbon evaporator. As with any technique there are innumerable perturbations and refinements; however the negative staining technique has consistently yielded high-resolution data on isolated dyneins obtained from Tetrahymena, Chlamydomonas , mammalian tracheal cilia, and Paramecium and on dynein-antibody and kinisin-microtubule complexes. There is a description of the method, preparation of mica, procedure, and precautions. Using a vacuum evaporator, mica sheets are coated with a thin layer of carbon. Once the carbon-coated mica has been prepared, it can be stored in a desiccator almost indefinitely. The 2- to 3-ml aliquot is carefully taken from below the surface of the stock solution, thus avoiding contaminants from precipitates that may exist at the surface and at the bottom of the stock solution. The precipitates themselves do not, in actual fact, affect the overall quality of the negative staining procedure, but they do result in a local heating of the support film when exposed to the electron beam, which, in its most benign form, can result in drift and at its worst will result in a ripping and curling of the support film. To avoid contamination, the tweezers or support grids are not to be dipped into either the protein solution or the negative staining solution. The dirt spread across the surface first and are preferentially bound, thus preventing the intact molecules that are in the bulk solution from binding moments later.