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上海交通大学:《生物化学 Biochemistry(B类)》课程教学资源(课件讲稿)Chapter 03 Exploring Proteins and Proteomes

1.The Purification of Proteins Is an Essential First Step in Understanding Their Function 2. The Characterization of Proteins: Molecular Weight, isoelectric point. 3 . Amino Acid Sequencing and primary structure
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上游文通大学 SHANGHAI JIAO TONG UNIVERSITY INTRODUCTION Protein (macromolecule,amino acid,peptide, structure,configuration,function) genome How to Explore? chromosomes .-----genes Genes contain instructions for making proteins Proteins act alone or in complexes to perform many cellular functions

Protein (macromolecule,amino acid,peptide, structure,configuration,function) INTRODUCTION How to Explore?

上游充通大学 SHANGHAI JIAO TONG UNIVERSITY INTRODUCTION Development: chip-based mass spectrometry approaches complex mixture analysis LC-MS(/MS) Proteins 2D gel electrophoresis proteomics 一time 1975 1990 1994 1996 2002 algorithms nucleotide sequencing Proteomes ESTs/genome scale genetic approaches

INTRODUCTION Development: Proteins Proteomes

上游充通大 OUTLINES SHANGHAI JIAO TONG UNIVERSITY Proteins 1.The Purification of Proteins Is an Essential First Step in Understanding Their Function 2.The Characterization of Proteins:Molecular Weight,isoelectric point. 3.Amino Acid Sequencing and primary structure

1.The Purification of Proteins Is an Essential First Step in Understanding Their Function 2. The Characterization of Proteins: Molecular Weight, isoelectric point. 3 . Amino Acid Sequencing and primary structure OUTLINES Proteins

上游充通大兽 OUTLINES SHANGHAI JIAO TONG UNIVERSITY Proteomes 4.Two-dimensional electrophoresis is currently the workhorse for the majority of ongoing proteome projects. 5.Mass Spectrometry Provides Powerful Tools for Protein Characterization and Identification 6.Three-Dimensional Protein Structures Can Be Determined by X-ray Crystallography and NMR Spectroscopy

4.Two-dimensional electrophoresis is currently the workhorse for the majority of ongoing proteome projects. 5. Mass Spectrometry Provides Powerful Tools for Protein Characterization and Identification 6.Three-Dimensional Protein Structures Can Be Determined by X-ray Crystallography and NMR Spectroscopy OUTLINES Proteomes

上游充通大学 OUTLINES SHANGHAI JIAO TONG UNIVERSITY Proteins 1.The Purification of Proteins Is an Essential First Step in Understanding Their Function 2.The Characterization of Proteins:Molecular Weight,isoelectric point. 3.Amino Acid Sequencing and primary structure

OUTLINES Proteins 1.The Purification of Proteins Is an Essential First Step in Understanding Their Function 2. The Characterization of Proteins: Molecular Weight, isoelectric point. 3 . Amino Acid Sequencing and primary structure

上游充通大学 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY 蛋白质的分离纯化: 复杂样品→目标蛋白 血红蛋白 红细胞 血液样本

1. The Purification of Proteins 血红蛋白 红细胞 血液样本 蛋白质的分离纯化: 复杂样品 目标蛋白

上游充通大兽 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY 蛋白分离纯化的目的与意义 获取高纯度蛋白:蛋白质修饰与蛋白结晶的前提 提高蛋白生化性能:排除杂蛋白的干扰 ©蛋白及多肽类药品质量要求:去除杂蛋白(抗原性) 兔 公 analysis

蛋白分离纯化的目的与意义 获取高纯度蛋白:蛋白质修饰与蛋白结晶的前提 提高蛋白生化性能:排除杂蛋白的干扰 蛋白及多肽类药品质量要求:去除杂蛋白(抗原性) 1. The Purification of Proteins

上游充通大学 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY 常用蛋白分离纯化方法 >根据分子大小、密度不同分离: 离心 透析 凝胶过滤层析 >根据分子电荷不同的分离方法:离子交换层析 >利用蛋白质亲和性差异进行分离:亲和层析 >蛋白质分离纯化系统:HPLC

1. The Purification of Proteins 常用蛋白分离纯化方法  根据分子大小、密度不同分离: • 离心 • 透析 • 凝胶过滤层析  根据分子电荷不同的分离方法:离子交换层析  利用蛋白质亲和性差异进行分离:亲和层析  蛋白质分离纯化系统:HPLC

上游充通大兽 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY 1)根据分子大小、密度不同的分离方法: a)Centrifugation:离心 血浆蛋白 7% 白蛋白54% 球蛋白38% 血浆 55% 纤维蛋白原7% 其它1% 水 91,5% 电解质 营养物质 气体 激素 血浆 推生素 55% 废物 其它物质 1,5% 血浆(重) 溶质 白国胞 血小板 中性粒细胞 口血小板 血细胞 459% 150,000-400.000 60-70% e991s 33 白细胞 5.000-10.000 红细鲍 45% 红细胞 淋巴细胞 4,8-5,4百万 20-25% 单核细胞 3-8% 血液样本 喷碱性粒细胞 全血 0.5-10% 嘴酸性粒细跑 2一4% 容量 有形成分 白细跑

1. The Purification of Proteins a) Centrifugation:离心 血液样本 1)根据分子大小、密度不同的分离方法:

上游充通大兽 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY Differential centrifugation:差速离心 利用样品中各种组分的沉降系数不同而进行分离的方法。在 差速离心中细胞器沉降的顺序依次为:细胞核、线粒体、微 粒体。 Centrifuge at5oo>×g for 10 minutes Supernatant Homogenate forms 10,000>×9 Pellet:Nuclear 20 minutes fraction 利用速度梯度,使 各种沉降系数不同 100,000>×g Pellet:Mitochondrial 的颗粒先后沉淀下 I hour fraction 来,达到分离的目 的。 Cytoplasm (soluble proteins) Pellet:Microsomal fraction

1. The Purification of Proteins Differential centrifugation:差速离心 利用样品中各种组分的沉降系数不同而进行分离的方法。在 差速离心中细胞器沉降的顺序依次为:细胞核、线粒体、微 粒体。 利用速度梯度,使 各种沉降系数不同 的颗粒先后沉淀下 来,达到分离的目 的

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