1 / 55 Lecture 29 Appendix D Experimental Techniques D.1 DNA manipulation techniques D.2 Manipulating Proteins D.3 The Function of Genes and their Proteins.

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1 1 / 55 Lecture 29 Appendix D Experimental Techniques D.1 DNA manipulation techniques D.2 Manipulating Proteins D.3 The Function of Genes and their Proteins 附录 D 实验技术 D.1 DNA 操作技术 D.2 操作蛋白质 D.3 基因及其 蛋白质的功能

2 2 / 55 D.2 Manipulating Proteins / 操作蛋白质 D.2.2 Protein Identification 蛋白质鉴定 D.2.1 Protein Purification and Separation 蛋白质纯化与分离

3 3 / 55 D.2.1 Protein Purification and Separation 蛋白质纯化与分离 1. Column Chromatography 柱层析 2. Protein Gels 蛋白质凝胶 3. Antibodies and Western Blotting 抗体与 Western 印迹法

4 4 / 55 1. Column Chromatography / 柱层析 Ion-exchange chromatography 离子交换层析 Gel-filtration chromatography 凝胶过滤层析 Affinity chromatography 亲和层析

5 5 / 55 Ion-exchange chromatography

6 6 / 55 Gel-filtration chromatography

7 7 / 55 Affinity chromatography

8 8 / 55 2. Protein Gels / 蛋白质凝胶

9 9 / 55 Protein Gels / 蛋白质凝胶

10 10 / 55 Now showing SDS gel electrophoresis File: biophoto7 \ Lehninger Principles of Biochemistry \ SDS gel electrophoresis (5’)

11 11 / 55 SDS gel electrophoresis (1/6) SDS-polyacrylamide gel electrophoresis is a powerful tool, which resolves proteins according to their molecular weights. Because proteins differ in size, shape, and charge, a protein sample is first denatured with the anionic detergents SDS.

12 12 / 55 SDS gel electrophoresis (2/6) When the sample is heated, the SDS molecules bind to the proteins and cause them to unfold. The denatured proteins become uniformly coated with negatively charged SDS molecules, so they all have a similar shape and charge-to-mass ratio.

13 13 / 55 SDS gel electrophoresis (3/6) If a protein is composed of several subunits, the SDS nor only unfolds the protein, but also dissociates the protein into its individual polypeptide chains.

14 14 / 55 SDS gel electrophoresis (4/6) The mixture of denatured proteins is then transferred from the tube and loaded into a well that has been cast in the top of a polyacrylamide gel. In an electric field generated by a power supply, the negatively charged polypeptides migrate through the gel toward the positive electrode at the bottom of the gel.

15 15 / 55 SDS gel electrophoresis (5/6) The migrating polypeptides are retarded by the tangled network of polyacrylamide. Smaller polypeptides travel more easily and quickly through the pores in the network than do larger polypeptides.

16 16 / 55 SDS gel electrophoresis (6/6) Because the polypeptides have similar charge-to- mass ratios, the distance they travel through a gel is dependent only on molecular weight. Based on this principle, proteins are separated according to their sizes, with low molecular weight proteins having greater mobility than high molecular weight proteins.

17 17 / 55 Now showing again

18 18 / 55 3. Antibodies and Western Blotting 抗体与 Western 印迹法

19 19 / 55 Western blotting / Western 印迹法

20 20 / 55 Now showing Immunoblotting File: biophoto7 \ Lehninger Principles of Biochemistry \ Immunoblotting (3’)

21 21 / 55 D.2.2 Protein Identification / 蛋白质鉴定 MALDI-TOF mass spectrometer (Medium Assisted Laser Desorption Ionization – Time Of Flight) 基质辅助激光脱附电离 - 飞行时间质谱仪

22 22 / 55 A example of mass spectrum 一个质谱的例子

23 23 / 55 Now showing --- 质谱仪工作站 File: 质谱仪工作站 (7’)

24 24 / 55 D.3 The Function of Genes and their Proteins / 基因及其蛋白质的功能 D.3.1 What is the protein’s structure? 蛋白质具有什么样的结构？ D.3.2 What Other Proteins is it related to? 它与其它哪些蛋白质有关？ D.3.3 When is the protein expressed? 这种蛋白质在什么时候表达？ D.3.4 When is the protein active? 这种蛋白质什么时候有活性？ D.3.5 Where is it expressed? 它在哪里表达？ D.3.6 What other proteins does it bind to? 它与其它哪些蛋白质结合？ D.3.7 What DNA sequences does it bind to? 它与什么样的 DNA 序列结合？ D.3.8 What happens if it is removed or altered? 如果它被移去或改变会怎样？

25 25 / 55 D.3.1 What is the protein’s structure? 蛋白质具有什么样的结构？

26 26 / 55 D.3.2 What Other Proteins is it related to? / 它与其它哪些蛋白质有关？

27 27 / 55 D.3.3 When is the protein expressed? 这种蛋白质在什么时候表达？

28 28 / 55 DNA microarray / DNA 芯片

29 29 / 55 DNA microarray / DNA 芯片

30 30 / 55 Now showing DNA Chip Technology File: biophoto7 \ Life \ DNA chip technology (5’)

31 31 / 55 Now showing Synthesizing an Oligonucleotide Array File: biophoto7 \ Lehninger Principles of Biochemistry \ Synthesizing an oligonucleotide array (3’)

32 32 / 55 D.3.4 When is the protein active? 这种蛋白质什么时候有活性？

33 33 / 55 Detection of protein modifications 蛋白质修饰的检测

34 34 / 55 Detection of protein modifications 蛋白质修饰的检测

35 35 / 55 D.3.5 Where is it expressed? 它在哪里表达？

36 36 / 55 Now showing Reporter Constructs File: biophoto7 \ Lehninger Principles of Biochemistry \ Reporter constructs (3’)

37 37 / 55 D.3.6 What other proteins does it bind to? / 它与其它哪些蛋白质结合？

38 38 / 55 A GST-pull down assay GST 下拉分析

39 39 / 55 The yeast two-hybrid / 酵母双杂交

40 40 / 55 Now showing Yeast two-Hybrid System File: biophoto7 \ Lehninger Principles of Biochemistry \ Yeast two-hybrid system (3’)

41 41 / 55 D.3.7 What DNA sequences does it bind to? 它与什么样的 DNA 序列结合？

42 42 / 55 Chromatin immunoprecipitation 染色质免疫沉淀法（ ChIP ）

43 43 / 55 The gel-shift assay / 凝胶移位分析

44 44 / 55 D.3.8 What happens if it is removed or altered? 如果它被移去或改变会怎样？

45 45 / 55 The gene knockdown / 基因敲低

46 46 / 55 Knockout mice / 基因敲除小鼠

47 47 / 55 Knockout mice / 基因敲除小鼠

48 48 / 55 Knockout mice / 基因敲除小鼠

49 49 / 55 Knockout mice / 基因敲除小鼠

50 50 / 55 Now showing Creating a Transgenic Mouse File: biophoto7 \ Lehninger Principles of Biochemistry \ Creating a transgenic mouse (3’)

51 51 / 55 Knockout mice → “ 大力 ” 鼠 www.mesomorphosis.com/articles/volk/myostatin.htm The Myostatin Gene

52 52 / 55 Knockout mice → “ 小胖 ” 鼠 Figure: p27 -/- mice (right) are heavier than their wildtype littermates (left) (www.fhcrc.org/.../fero/RL_gifs/Mouse%20scale.jpg)

53 53 / 55 Knockout mice → “ 大胖 ” 鼠 www.umass.edu/neuro/faculty/files/good.htm Deborah J. Good Nhlh2 knockout mice (N2KO)

54 54 / 55 Knockout mice → “ 矮脚 ” 鼠 Three mice that are littermates (siblings of the same age). The two mice on the left (one partially hiding under the big mouse) are dwarf mice caused by the Perk mutation. They are less than half the size of the mouse on the right, which is normal. Micro x-ray computed tomography (microCT) images of the tibia of normal and Perk mutant mice. The mutant shows thin cortical bone with deformations and gaping holes. "If it wasn't for muscle and connective tissue to hold these fragile bones together, these mice would fall apart," Cavener says. PERK (pancreatic endoplasmic reticulum kinase)

55 55 / 55 Double Knockouts 双敲鼠 Plasminogen （血纤蛋白溶酶原） deficient mice (Plg-/-) vs double knockout mice, note the severe growth retardation corrected by fibrinogen （血纤蛋白原） knockout. (www.talkreason.org/img/clots/image001.jpg )