杏树皮可溶性总蛋白的提取与浓缩方法探讨

Study on the Extracting and Condensing Methods of Total Proteins from the Shoot Barks of Apricot

为从杏树皮组织中获取高质量的可溶性总蛋白,选择经过冬季自然低温驯化的1 a生杏树新梢为试材,比较分析提取缓冲液中分别添加2%SDS,不同浓度的Tween 20,以及高浓度PVPP对可溶性总蛋白提取浓度和质量的影响。结果表明:提取杏树皮可溶性总蛋白的最佳缓冲液体系为:5 mL 50 mmol/L的Tris-HCl缓冲液(其中含2 mM EDTA-Na2,10 mmol/L抗坏血酸,0.5 mmol/L DTT,0.1 mmol/L PMSF,1%(w/v)PVPP,pH 8.0)和0.5%Tween20。丙酮浓缩方法中加入蛋白粗提液2倍体积的冷丙酮是最理想的。经过提取和浓缩的蛋白浓度可达0.773μg/μL,电泳分析中的条带多而清晰,可用于后续的目的蛋白纯化试验及功能分析。

The condition of extracting soluble total proteins from the shoot barks of apricot by different solvents was discussed.The optimum extracting condition was determined as：50 mmol/L Tris-HCl buffer,pH 8.0,containing 2 mmol/L EDTA-Na2,10 mmol/L ascorbic acid,0.5 mmol/L DTT,0.1 mmol/L PMSF,1%（w/v） PVPP and 0.5%（w/v） Tween20.The acetone condensing method was also investigated and the results showed that the volume ratio of the rude protein extracts to acetone was 1∶2,the reacting result was most desirable： the protein concentration was higher to 0. 773μg/μL, and many clear bands was detected by SDS-PAGE, which indicating that the protein extracts had a good quality for deeply study, such as protein purification and function analysis.