【摘要】 目的通过对蛋白质样本制备及IEF蛋白质上样时几个重要环节进行探讨,为乳腺癌组织蛋白质样本2-DE分离结果优化提供重要的实验与理论的依据。方法通过Bio-Rad公司提供的试剂盒对乳腺癌组织进行总蛋白提取、蛋白质纯化、蛋白质定量,并分别对处理前后的乳腺癌蛋白质样本进行SDS-PAGE与2-DE分离。结果乳腺癌组织蛋白质的等电点主要集中在pI5~8范围内,SDS-PAGE、蛋白质样本纯化与定量等措施及适当的IEF上样体积与上样量可获得良好的2-DE蛋白质分离结果。结论样本制备是2-DE蛋白质分离的关键,准确的取材、蛋白质样本的SDS-PAGE质量监测、适量的样本上样体积与上样量、适当的pH值范围的IPG胶条的选择以及采用蛋白质纯化试剂盒对蛋白质样本进行纯化与浓缩是整个实验成功的保证。更多还原

【Abstract】 Objective To supply important experimental and theoretical data for good results of separating of protein by 2-DE, some operation about preparation of protein sample and loading of protein sample in IEF were explored. Methods Extraction of total protein from breast cancer tissue and purification and quantitation of them were done with corresponding kits provided by Bio-Rad, then before and after being treated thses samples of proteins were respectively separated by SDS-PAGE and 2-DE. Results Good experimental results were obtained by selection of IPG strip with pH5-8, SDS-PAGE observation of raw proteins, purification and quantitation of total proteins and adequately loading volume and content of sample in IEF. Conclusion Preparation of protein sample is the key in 2-DE, and some operation are also very important factors which interfere with experimental results, including correct materials drawn, quality control of sample by SDS-PAGE, proper loading volume and content of sample in IEF, adequately loading volume and content of sample in IEF, selection of IPG strip with proper range of pH valve, as well as use of Cleanup Kit for purification and concentration of sample.更多还原