Extraction Method of Total Protein from Trichothecium roseum Suitable for Proteomic Analysis

doi:10.7668/hbnxb.201751366

Abstract

Abstract: In order to optimize the protein extraction method suitable for two-dimensional electrophoresis of Trichothecium roseum and further study the pathogenic mechanism of T. roseum on the proteomic level, the isolated and purified T. roseum were chosen to extract the total protein by 5 methods:ultrasonic-TCA-acetone, ultrasonic-phosphoric acid-TCA acetone, ultrasonic-SDS, ultrasonic-TCA/acetone-phenol/SDS combined extraction and TCA/acetone-phenol/SDS combined extraction,respectively. Meanwhile, two better extraction methods were screened by comparative analysis of protein content and purity. Under the appropriate gel concentration of polyacrylamide electrophoresis, the best protein extraction method was obtained by two-dimensional electrophoresis. The results showed that 12% polyacrylamide gel concentration had clear background and no serious tailing. Moreover, the concentration and content of protein samples obtained by the method of ultrasonic-TCA/acetone-phenol/SDS combined extraction were 6.650 mg/mL and 2.993 mg/g respectively. Furthermore, clear bands were obtained in the extracted protein by SDS-PAGE analysis and 1 238 independent clear protein spots were obtained by two-dimensional electrophoresis analysis. It is suggested that the T. roseum protein obtained by the combined extraction method of ultrasonic-TCA/acetone-phenol/SDS was suitable for two-dimensional electrophoresis analysis and further proteomic research.

Key words: Trichothecium roseum, Proteome, Protein extraction, Two-dimensional electrophoresis

CLC Number:

S432.1

WANG Peng, WANG Junjie, LI Zhenbiao. Extraction Method of Total Protein from Trichothecium roseum Suitable for Proteomic Analysis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(3): 209-216. doi: 10.7668/hbnxb.201751366.

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