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Agarose Gel Electrophoretic pH-Mobility (Titration) Curves of Isometric Plant Viruses. S. S. Hurtt, Agricultural Research Service, U.S. Department of Agriculture, National Plant Germplasm Quarantine Laboratory, Bldg. 580, BARC-East, Beltsville, MD 20705; D. Tietz, J. S. Fawcett, and A. Chrambach. Section on Macromolecular Analysis, Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892. Phytopathology 79:661-667. Accepted for publication 4 January 1989. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1989. DOI: 10.1094/Phyto-79-661.

Electrophoretic pH-mobility curves (also known as titration curves) were used to analyze virions of six purified isometric plant viruses. Curves were generated in agarose slab gels composed of 0.8% IsoGel agarose, 10% d-sorbitol, and 2% carrier ampholyte. In the first dimension of this two-dimensional electrophoretic procedure, the carrier ampholytes were focused to a steady state, creating a pH gradient in the gel. Virus was applied in a continuous band across the pH gradient, and an electrical field was applied to the prefocused gel at right angles to the first field. This step showed the virion mobility over a range of pH values. Protein was fixed and stained to visualize the curve. Each virus gave a unique curve. The procedure established the isoelectric point (pI) of the virus and the virion mobility (that is, net surface charge) as a function of pH in a single gel experiment. Previously, these data could only be obtained by conducting numerous electrophoretic experiments. Curves also revealed virion pH instabilities and heterogeneity within preparations. Curves were standardized using a computer program to normalize nonlinear variations in the pH gradient within a gel and among gels.

Additional keywords: cucumber mosaic virus, hibiscus chlorotic ringspot virus, pelargonium flowerbreak virus, tomato bushy stunt virus, turnip crinkle virus, two-dimensional isoelectric focusing-gel electrophoresis.