摘要: 本课题通过建立尿液总蛋白的提取方法并进行优化，为深入开展尿液蛋白质组学研究提供基础。本课题采用目前最常用的两种方法（丙酮沉淀法和超滤法），分别提取尿液总蛋白后进行分析比较，发现两种方法各有优缺点。丙酮沉淀法提取的总蛋白得率高，且操作简便、成本低廉，但电泳条带较单一；超滤法提取的总蛋白电泳条带更丰富，但耗时长，成本较高；因此我们尝试针对丙酮沉淀法进行优化改良，以达到甚至超过超滤法的目的，从而节约成本和时间。首先针对丙酮沉淀后采用不同裂解液进行溶解提取，发现对于电泳结果并无影响；进一步，针对不同的沉淀剂进行了对比，结果发现TCA/丙酮作为沉淀剂，可以有效去除尿液白蛋白高丰度蛋白干扰，从而显著提高蛋白条带的丰富性，但其缺点是得率较低。然而，尿液作为一种极易获取的临床样本，提高尿液量即可弥补得率的损失。因此，本课题通过综合比较后确定TCA/丙酮法是最合适尿液总蛋白提取的方法。81213

毕业论文关键词：尿液；蛋白质组学；方法优化；TCA/丙酮

Establishment and optimization of urinary total protein extraction methods

Abstract: In this study, we established the method of extracting urine total protein and optimized it, which provided the basis for further study of urine proteomics。 In this study, the two most commonly used methods (acetone precipitation and ultrafiltration) were used to analyze the total protein of urine, and it was found that the two methods had their own advantages and disadvantages。 The total protein yield extracted by acetone precipitation method is high, and the operation is simple and the cost is low, but the electrophoretic band is single。 The total protein electrophoresis band extracted by ultrafiltration was richer, but it was time consuming long and costly。 Therefore, we try to optimize the acetone precipitation method to achieve or even exceed ultrafiltration method, to save costs and time。 Firstly, the results showed that there was no effect on the electrophoretic results when the urine protein precipitated by acetone and dissolved by different lysates。 Furthermore, compared with different precipitants, it was found that TCA/acetone as a precipitant could effectively remove the high abundance protein of urine albumin, which could significantly improve the protein band, but the disadvantage was that the yield low。 However, urine as a very easy access to clinical samples, increase the amount of urine can make up for the loss of yield。 Therefore, this study through the comprehensive comparison to determine TCA/acetone method is the most appropriate method of total protein extraction of urine。

Key Words: urine; proteomics; method optimization; TCA/acetone

目  录

1 绪论 1

1。1 蛋白质 1

1。2 蛋白质组学 1

1。3 尿液蛋白质组学 2

1。3。1 尿液 2

1。3。2 尿蛋白 2

1。3。3 尿液蛋白质组学研究现状 2

2 本课题的研究目的和意义 4

2。1 本课题的研究目的 4

2。2 本课题的研究意义 4

3 实验部分 5

3。1 实验试剂 5

3。2 实验仪器设备 6

3。3 所用试剂以及试剂配制 7

3。3。1 8M尿素+1‰SDS裂解液