Preparation of an Antiserum against an in vitro Translation Product of Alfalfa Mosaic Virus RNA

Antisera were raised against P3 (mol. wt. 32 000), the full-length translation product of alfalfa mosaic virus (A1MV) RNA 3. P3 was made by translation in wheat germ extracts, using unfractionated AlMV RNA as message, and the products were then fractionated either by centrifugation through a sucrose gradient, or by phosphocellulose chromatography, each technique being followed by SDS-PAGE. Preparations made by each method were used in sequence to immunize a rabbit. The resulting antisera reacted on immunoblots with P3 synthesized in vitro, at dilutions of about 1 : 10000, but did not react with the translation products of A1MV RNA 1, RNA 2 or RNA 4. Although the antisera contained antibodies against some wheat germ components, most were removed by preabsorbing the antisera with the wheat germ extract. A minor wheat germ translation product of RNA 3, P'3, also reacted with the antisera. Since the mol. wt. of P'3 is apparently 1500 higher than that of P3, it is possible that P'3 is a readthrough product of the P3 cistron. With one antiserum preparation we were able to detect P3 in extracts of tobacco plants infected with AIMV.