生化与分子生物学实验原理I2011_绪论

生物化学与分子生物学实验原理(I) 熊英李旭李卫芳朱中良吴高等2011年9月06日1、教学大纲和课程安排2、常用网址3、简要历史4、引子第二章蛋白的分离纯化方法（李旭）第三章蛋白纯度鉴定，分子量及浓度测定（李卫芳）第四章毛细管电泳和蛋白质N端测序技术（施荣华）第五章蛋白-蛋白相互作用研究方法的原理简介（张海燕）第六章光谱学原理及其在生物学中的应用（周宏敏）第七章气相，液相质谱技术（熊英，吴高）第八章蛋白质翻译后修饰的生化研究原理及方法（朱中良）2011-09-06:第一章绪论(熊英)2011-09-13:第二章蛋白的分离纯化方法(李旭)2011-09-20:第二章蛋白的分离纯化方法(李旭)2011-09-27:第三章蛋白纯度鉴定及分子量及浓度测定(李卫芳) 2011-10-11:第三章蛋白纯度鉴定及分子量及浓度测定(李卫芳) 2011-10-18:第四章毛细管电泳和蛋白质N端测序技术(施荣华)2011-10-25:第五章蛋白-蛋白相互作用研究方法的原理简介(张海燕）2011-11-01:第六章光谱学原理及其在生物学中的应用（周宏敏）2011-11-08:第七章气相质谱技术（熊英）2011-11-15：第七章液相质谱技术（吴高）2011-11-22：第七章液相质谱技术应用（熊英）2011-11-29:第八章蛋白质翻译后修饰的生化研究原理及方法(朱中良) 2011-12-06:第八章蛋白质翻译后修饰的生化研究原理及方法(朱中良)2011-12-09至2011-12-30 第九章实验课每周二四，14:00-16:25, 与《细胞生物学实验方法与原理》合并：每位同学任选以下2项技术（1项主选，1项辅选）进入相应仪器室跟随指导老师实习，并完成实习报告，总学时不得少于3X4=12学时；2012-01-03 考试（笔试70分，实验30分）时间14:00-16:001.Biacore技术（欧惠超）2.化学发光与生物发光检测技术（罗昭锋）3.激光共聚焦显微镜技术（主辅修人数各<28人）（吴旭）4. 流式细胞术（主辅修人数各<20人）（王黎丽）5. 活细胞工作站技术（主辅修人数各<20人）（刘雅静）6. 液质联用技术（吴高）7. 气质联用技术（熊英）8. 毛细管电泳及蛋白质测序技术（施荣华）9. 荧光定量PCR技术（张海燕）10. 离心技术(王昊)11. HPLC及蛋白质纯化技术（周宏敏）联系方式：李旭sachem@ 李卫芳liwf@张海燕zhy2009@ 施荣华rhsh@周宏敏itisme@ 吴高wugao@ 朱中良zlzhu63@ 欧惠超ohc@罗昭锋lzf@吴旭wuxu@王黎丽wlili@刘雅静yjl@王昊hwang@课件下载地址：教务处---精品课程---省级精品课程---生物化学---实验课程文档/webapps/portal/frameset.jsp?tab_i d=_2_1&url=/bin/common/course.pl?course_id%3D_138_1联系方式：熊英生命学院336房间电话：3600425yxiong73@1、教学大纲和课程安排2、常用网址3、简要历史4、引子Useful websites:1, National Center for Biotechnology Information (NCBI) /Pubmed, Entrez, Blast, …2, The ExPASy(Expert Protein Analysis System):/3, IMB-Jena Amino Acid Repository:http://www.imb-jena.de/IMAGE.htmlHelpful websites:1, PDB (Protein Data Bank)/pdb/home/home.do2, SCOP (Structural Classification of Proteins)/scop/3, comparison of protein structures in 3D /dali/index.html1、教学大纲和课程安排2、重要网址3、简要历史4、引子and genetics.1835 Jons Berzeliuschemical catalysis, uses amylase(淀粉酶) as an example 1859 Charles Darwin publishesOn the Origin of Species.1860 Louis Pasteur recognizesfermation catalyzed by enzymes, "essence" of yeast. 1865 Gregor Mendel publishestheory of genetics.1869 Fredrick Meischer discoversDNA in cell nuclei.1897 Eduard and Hans Buchner extracts materiel from yeast, conversion of glucose to alcohol.1914 Fritz Lipmann elucidatesthe role of ATP in energy metabolism.1926 James Sumner obtainscrystalline jack bean (刀豆) urease, is a protein. 1926 Thomas Hunt Morgan writesThe Theory of the Gene.1934 Arnold Beckman developesthe first pH meter.1937 Hans Krebs discoversthe citric acid cycle(TCA cycle).1941 George Beadle & Edward Tatum proposethe one-gene, one-enzyme hypothesis.Oswald Avery, Colin MacLeod, and Maclyn McCarthy DNA is the genetic material.1950 Edwin ChargaffA=T, G=C(Chargaff's rules).1952 Linus Pauling and Robert Corey proposeα-helix and the β-pleated sheet1953 James Watson and Frances Crickthe double helix model of DNA.1953 Fredrick Sanger determinesthe first amino acid sequence of a protein(insulin). 1956 Earl Sutherland isolatescyclic AMP.1957 Matthew Meselson and Franklin Stahl demonstrate semiconservative DNA replication.1960 John Kendrew and Max Pertuz obtain the first 3-D structure of proteins(hemoglobin and myoglobin). 1960 Jerald Huritz and Samuel Weiss discoverRNA polymerase.1961 Francois Jacob and Jaques Monod propound the operon model of gene control.1963 Jean-Pierrre Changuex, F. Jacob, and J. Monod Allosteric model for inhibition of enzymes1964 Several groupsAcrylamide gel electrophoresis of proteins is developedMarshal Nirenberg, H. Gobind Khorana, and Severo Ochoa complete the elucidation of the genetic code1965 David Phillips3-D model of first enzyme(lysozyme)1965 Robert Holley determinesthe structure of a transfer-aaRNA.1965 Jerome Vinograd discoverssuperhelical twisting.1968 Mark Ptashne and Walter Gilbert identifythe first repressor genes1969First synthesis of an enzyme(ribonuclease).1970 Hamilton Smith discoversrestriction endonucleases.1970 Howard Temin and David Baltimore discoverreverse transcriptase1973 Stanley Cohen and Herbert Boyer preparerecombinant DNA.1974 Sung-Hou Kim, et al. producethe first X-ray structure of transfer RNA.1977 Cesar Milstein discovers how to producemonoclonal antibodies.1977 Allan Maxam and Walter Gilbert developa chemistry for sequencing DNA.1977 Fredrick Sanger, S. Nicklen and A.R. Coulson developa chemistry for sequencing DNA.1977 Phillip Sharp and Richard Roberts discoverintrons(intervening sequences).1982 Amzel LM, McKinney M, Narayanan P, Pedersen PL First x-ray structure of a membrane protein (9Å).The era of Omics: (1998-2003 or later?) Omics= Oh! Mix!1、教学大纲和课程安排2、重要网址3、简要历史4、引子Central Dogma (Crick F.1958)Molecular Biology Human Genome Project (1990-2003)Genomics Omics:ProteomicsTranscriptomicsMetabolomicsStructural Genomics(1998-)1, Targets selection: Bioinformatics 2, PCR and cloning: MolecularBiology3, Protein production: Biochemistry 4, Data collection (X-ray/NMR)5,Structure determination:Structural Biology6, Function interpretation:General BiologyFlow chartCore characteristicsHigh throughput(hundreds to thousands samples)Large-scale(milligram level of protein sample)Multi-discipline integration(from molecular biology to structural biology)Proteins samples for crystallization trials:1, pure and homogenous2, stable in solution of low salt concentration, proper pH and reductant (BME, DTT or TCEP) 3, at high concentration (normally ~10 mg/mL)Optimization of xtals:1, about half of the xtals will not diffract at 3Åor higher resolution2, parameters: salt concentration, buffer system, pH, divalent or trivalent irons3, precipitants: PEGs, MPD, salt, alcohol, …4, ligands or substrates/products/analogs5, protein or nucleic acid partnersin-house X-raygeneratorData collection:synchrotron radiationaccelerator 追寻隐形的光线——X 射线源发展小史Function interpretation and application:•Assignment of function based on structural similarity •Reciprocal stimulation and validation: biochemical assays versus structure analyses•Structure-directed drug design。