Physical Principles and Techniques of Protein Chemistry by Sydney Leach

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By Sydney Leach

Actual ideas and methods of Protein Chemistry half A.

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SLAYTER FIG. 7a. Glycogen from Entamoeba mvadens, negatively contrasted by uranyl acetate. The large, irregular, relatively electron-transparent glycogen particles are outlined by the electron-opaque stain. e. more than about 1 hour) or occasionally, to unpredictable effects of atmospheric conditions. Treatment with vapors of hydrofluoric or hydrochloric acid may facilitate stripping, but only at the risk of damage to the specimen. B. NEGATIVE CONTRAST 1. Method The method of negative contrast (commonly, if rather inaccurately, called negative staining) consists of embedding relatively electron-transparent particles in a continuous electron-opaque film.

The fractured specimen in its frozen state is subjected to a vacuum which causes partial freezedrying. In this way, moisture is sublimed from the surface layers of the fracture, exposing the tissue (or molecules) in their native configuration. A replica of the surface may then be made and observed in the microscope. Since the freeze-drying of the frozen block is incomplete, sublimation of macromolecules should not be a problem. F. T H E CRITICAL-POINT TECHNIQUE The collapse of particles which is observed to occur during air drying may be attributed to the effect of surface tension during the abrupt phase changes which occur as the solvent evaporates.

A mica layer sufficiently thick for convenient handling would be electron-opaque, but the shadow replica, together with the supporting layers, can be removed by flotation and viewed in the electron microscope. Practical difficulties with the shadow-transfer technique are usually concerned with the process of stripping the shadowing layer from the mica. Water must be allowed to penetrate between the hydrophilic mica and the metal. Any failure in the hydrophilic character of the surface will prevent this; hence, the importance of using freshly cleaved mica.

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