The use of SDS-polyacrylamide gel electrophoresis to assess purification procedures of three potyviruses infecting cucurbitaceous plants.

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  • SUZUKI Nobuhiro
    Faculty of Agriculture, Tohoku University Laboratory of Plant Genetic Engineering, Biotechnology Institute, Akita Prefectural College of Agriculture
  • SHIRAKO Yukio
    Faculty of Agriculture, Tohoku University Center for Agricultural Molecular Biology, Cook College, Rutgers University
  • EHARA Yoshio
    Faculty of Agriculture, Tohoku University

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  • SDS-PAGEによるウリ科植物感染性potyvirus3種の精製法の検討
  • SDS-PAGEによるウリ科植物感染性potyvirus3種の精製法の検討〔英文〕
  • SDS-PAGE ニ ヨル ウリカ ショクブツ カンセンセイ potyviru

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Abstract

We examined the effects of concentration methods, extraction buffers, clarification reagents, and resuspension buffers on virus yield and purity by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), so as to establish optimal purification procedures of zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus 2 (WMV2), and the watermelon mosaic virus 1 strain of papaya ringspot virus (PRSV-W). We then investigated the physicochemical properties of the three viruses. SDS-PAGE proved very useful to monitor virus purification in a small scale. The three viruses were purified with similar yields of 5-10mg per 100g of infected pumpkin leaves by the respective optimal methods. The procedures basically involved clarification with Triton X-100, differential centrifugation using a 20% sucrose pad, and 20-50% sucrose density gradient centrifugation. From WMV2 and PRSV-W purified preparations, capsid protein (CP) of 34, 000 (34K) was predominantly detected in addition to minor degradation products of 27K to 33K, respectively. A breakdown product of 28K was, however, as detectable as intact CP of 33K from ZYMV purified preparation. The RNAs of WMV2 and PRSV-W, which were a little larger than ZYMV RNA, were indistinguishable in size as evaluated with 1.7% agarose gel electrophoresis in denaturing conditions.

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