Elsevier

Methods in Enzymology

Volume 96, 1983, Pages 466-485
Methods in Enzymology

[37] Immunoelectron microscopy using thin, frozen sections: Application to studies of the intracellular transport of Semliki Forest virus spike glycoproteins

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Publisher Summary

This chapter describes the practice and application of the thin frozen section technique to the biogenesis of the semliki forest virus (SFV) membrane glycoproteins, and presents a general procedure for making high-titered antiserum to amphiphilic membrane proteins. The results indicate that the spike proteins pass through the Golgi stacks, and antibodies to the spike proteins specifically label all the membrane compartments on the intracellular transport pathway. Parallel biochemical experiments show that the spike proteins acquired complex sugars in the Golgi stacks, but the precise location is not determined. The spike proteins move from cis to trans Golgi cisternae. The spike proteins pass through the Golgi stacks during intracellular transport and are present in all cisternae of the stack. The concentration of spike proteins in endoplasmic reticulum (ER) and Golgi membranes is extremely low. Radiolabeling infected cells with defined specific-activity amino acids show that a typical infected cell, 4-6 hr after infection, is synthesizing about 120,000 spike proteins per minute.

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