蛋白质相关实验技术操作指南

1、1、Western blotting:Western blotting using ECL reagent used by：Laboratory of P.J. Hansen,Dept. of Animal Sciences, University of Florida Kampss Western Blotting Protocolused by：Sefton Lab, Salk InstituteDry Transfer 干法蛋白转膜used by：The Preuss Lab,The Division of Biological Sciences,The University of Ch

2、icago Western Blot of TBP from TBP-GST bacteriaused by：Steven Finkbeiner, Departments of Neurology and Physiology, UCSFWestern Blot/Anti-P-CREBused by：Steven Finkbeiner, Departments of Neurology and Physiology, UCSFWestern blotting protocol for 1C2, 3B5H10, and 4C8 Antibodiesused by：Steven Finkbeine

3、r, Departments of Neurology and Physiology, UCSFProtocol for anti-HA antibody Western Blottingused by：Steven Finkbeiner, Departments of Neurology and Physiology, UCSFProbing and Stripping of Western Blotused by：Hahn Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteWe

4、stern Blotting Protocolused by：Mirmira Laboratory at the University of VirginiaWestern Blotting and Immunostainingused by：Chazin Lab, Center for Structural Biology, Vanderbilt University Western Blotsused by：Lab of Dr. Mark Barton Frank, Oklahoma Medical Research FoundationWestern Blotting - Antibod

5、iesused by：The Minion Lab, College of Veterinary Medicine at Iowa State UniversityWestern Blottingused by：Cepko/Tabin Lab, Harvard UniversityCarbonate Solution For Western Blotused by：Dr. DE Koshland, Carnegie Institution of WashingtonWestern Blot with BSAused by：Dr. DE Koshland, Carnegie Institutio

6、n of WashingtonMETHOD for Western Blotsused by：Dr. DE Koshland, Carnegie Institution of WashingtonWestern Blotsused by：Michael Koelles Lab,Department of Molecular Biophysics and BiochemistryYale University2、蛋白分离/提取/浓缩 Lysis Buffersused by：Steven Finkbeiner, Departments of Neurology and Physiology, U

7、CSFYeast Protein Isolation Yaffe-Schatzused by：Amberg Lab ,Upstate Medical UniversityGuanidine Hydrochloride Purification of Proteins From SDS PAGEused by：Gottschling Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteYeast protein prep for SDS PAGE and western (rapid)

8、used by：Gottschling Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteYeast protein prep for SDS PAGE and western (glass bead)used by：Hahn Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstitutePurification of TFIIIC from yeastused by：Hahn L

9、ab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstitutePurification of TFIIA from E. coliused by：Hahn Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteTCA protein precipitationused by：Hahn Lab，The Fred Hutchinson Cancer Research Center and H

10、oward Hughes Medical InstitutePurification of MLCK ( Myosin Light Chain Kinase )from Rabbit Skeletal Muscle肌球蛋白轻链激酶used by：Stull Lab,University of Texas, Southwestern Medical Center Purification of Calmodulin from Bovine Brainused by：Stull Lab,University of Texas, Southwestern Medical CenterMyosin L

11、ight Chain Preparation from Skeletal and Cardiac Muscleused by：Stull Lab,University of Texas, Southwestern Medical CenterPurifying Protein from Inclusion Bodiesused by：Chazin Lab, Center for Structural Biology, Vanderbilt UniversityProtocol for Protein Extraction from Plantused by：The University of

12、Nebraska-Lincoln Protein Core Facility (PCF)Taq Polymerase Purificationused by：Cepko/Tabin Lab, Harvard UniversityYeast Protein Prep (No Boil)used by：Dr. DE Koshland, Carnegie Institution of WashingtonYeast Protein Prep (Boil)used by：Dr. DE Koshland, Carnegie Institution of WashingtonGST Fusion Prot

13、ein Prepused by：Vesicle Trafficking, Stanford University3、蛋白质检测分析：AMIDO BLACK STAINused by：Hancock Laboratory Methods,Department of Microbiology and Immunology,University of British Columbia, British Columbia, CanadaJames Hardwicks angiotensin assay protocolused by：Sefton Lab,Salk Institute Phosphoa

14、mino acid analysis:Mark Kampss methodused by：Sefton Lab,Salk InstituteULTIMATE HIS-UB ASSAY FORused by：the Tansey Lab at Cold Spring Harbor LaboratoryULTIMATE HIS-UB ASSAY FOR YEASTused by：the Tansey Lab at Cold Spring Harbor LaboratoryProtocol for Beta-Hexosaminidase RBL-2H3 Secretion Assayused by：

15、meyer lab,Stanford UniversityMeasurement of Myosin Regulatory Light Chain (RLC) Phosphorylationused by：Stull Lab,University of Texas, Southwestern Medical CenterBicinchoninic Acid (BCA) Protein Assayused by：David R. Caprette, Rice UniversityBiuret Protein Assayused by：David R. Caprette, Rice Univers

16、ityBradford protein assayused by：David R. Caprette, Rice UniversityHartree-Lowry and Modified Lowry Protein Assaysused by：David R. Caprette, Rice UniversityColorimetric Assaysused by：David R. Caprette, Rice UniversityKd measurement, fitting, calculation, and simulationused by：Chazin Lab, Center for

17、Structural Biology, Vanderbilt University4、蛋白标记修饰DSP Crosslinkingused by：Peter Novick Lab, Department of Cell Biology Yale University School of MedicineLabeling Gc-Globulin with Carboxyfluorescein Succinimidyl Esterused by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)Labeling Tubu

18、lin with Tetramethylrhodamine Succinimidyl Esterused by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)Labeling Gizzard Vinculin with Tetramethylrhodamine Iodoacetamideused by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)Labeling Gizzard Alpha-actinin with Tetrame

19、thylrhodamine Iodoacetamideused by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)Labeling Gizzard Myosin with Tetramethylrhodamine Iodoacetamide (primarily 17kD MLC)used by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)Labeling Gizzard Myosin with Tetramethyorhoda

20、mine Iodoacetamide (MHC and 17kD MLC)used by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)Labeling Muscle Actin with N-(1-pyrene) Iodoacetamideused by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)Labeling Muscle Actin with Tetramethylrhodamine Iodoacetamideused

21、by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS) Labeling Muscle Actin with Carboxyfluorescein Succinimidyle Esterused by：Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)Labeling Muscle Actin with 5-Iodoacetamidofluoresceinused by：Yu-Li Wang Lab,University of massa

22、chusetts Medical School(UMASS)ultimate mammalian cell pulse-chaseused by：the Tansey Lab at Cold Spring Harbor Laboratoryultimate yeast pulse-chaseused by：the Tansey Lab at Cold Spring Harbor LaboratoryGluteraldehyde Conjugation of Oligopeptides to Proteinsused by：Lab of Dr. Mark Barton Frank, Oklaho

23、ma Medical Research FoundationFluorescein labeling of proteinsused by：David Chambers,Salk instituteBiosynthetic labelingused by：Sefton Lab, Salk Institute , San Diego, California 5、免疫沉淀(IP/CoIP/ChIP) Immunoprecipitation buffers and protocolsused by：Steven Finkbeiner, Departments of Neurology and Phy

24、siology, UCSFCell Lysis/Western/IPused by：Steven Finkbeiner, Departments of Neurology and Physiology, UCSFAmplification and Labelling with Cy dyesused by：Yale Genome Analysis Center, Yale UniversityChromatin Immunoprecipitation from Mammalian Cell Extractsused by：Yale Genome Analysis Center, Yale Un

25、iversityChromatin Immunoprecipitation from Yeast Whole Cell Extractsused by：Yale Genome Analysis Center, Yale UniversityImmunoprecipitationused by：Hancock Laboratory Methods. Department of Microbiology and Immunology,University of British Columbia, British Columbia, CanadaUltimate yeast denaturing I

26、Pused by：the Tansey Lab at Cold Spring Harbor LaboratoryULTIMATE FREEZE-THAW LYSIS FORused by：the Tansey Lab at Cold Spring Harbor LaboratoryUltimate mammalian nondenaturing IPused by：the Tansey Lab at Cold Spring Harbor LaboratoryUltimate mammalian denaturing IPused by：the Tansey Lab at Cold Spring

27、 Harbor Laboratory Ultimate yeast ChIP assayused by：the Tansey Lab at Cold Spring Harbor Laborat oryChromatin Immunoprecipitation Assay and PCRused by：Hahn Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteYeast Chromatin Immunoprecipitation (ChIP)used by：Hahn Lab，The

28、 Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteMirmira Lab ChIP Protocolused by：Mirmira Laboratory at the University of VirginiaChIP Assay Protocolused by：Mirmira Laboratory at the University of VirginiaImmunoprecipitation and Immune Complex kinase assayused by：Sefton Lab

29、, Salk InstituteGeneral Principles of Immunoprecipitationused by：Sefton Lab, Salk Institute6、 融合蛋白表达及纯化 Use of B-PER lysis reagent to purify recombinant proteins tagged with His6 or maltose binding proteinused by：Smith Lab,Division of Biological Sciences,University of Missouri Bacterial Expression o

30、f Kinesin Motorsused by：Liz Greene and Steve HenikoffPurification of recombinant sBRF M166Lused by：Hahn Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteProtein Expression and Purification of RPA70-ABused by：Chazin Lab, Center for Structural Biology, Vanderbilt Unive

31、rsityCo-Expression and purification of human DNA primase p49-p58 subunits引发酶p49-p58亚基共表达纯化方法used by：Chazin Lab, Center for Structural Biology, Vanderbilt UniversityProtein Expression and Purification of MBP-T antigen MBP-T抗原表达纯化used by：Chazin Lab, Center for Structural Biology, Vanderbilt University

32、 Perspectives on Baculovirus Expression Systems表达used by：Lab of Dr. Mark Barton Frank, Oklahoma Medical Research FoundationExpression & Purification of His Tagged Proteins in E. coliused by：Cepko/Tabin Lab, Harvard University7、相互作用分析 Amplifying a large phage-display library without losing diversityu

33、sed by：Smith Lab,Division of Biological Sciences,University of MissouriProtein Interaction Analysis Using an IASYS Biosensorused by：Kitto Lab, The University of Texas at Austin Yeast Two-Hybrid Screen with Library and Baitused by：Mirmira Laboratory at the University of Virginia8、蛋白电泳/双向电泳 SDS-PAGEus

34、ed by：Steven Finkbeiner, Departments of Neurology and Physiology, UCSFGel Drying干胶used by：Steven Finkbeiner, Departments of Neurology and Physiology, UCSFNuPAGE Gel Electrophoresis 蛋白电泳used by：Kitto Lab, The University of Texas at AustinIsoelectric Focussing of Membrane Proteins by Slab Gel Methodus

35、ed by：Hancock Laboratory Methods,Department of Microbiology and Immunology,University of British Columbia, British Columbia, CanadaSilver Staining SDS PAGE Gelsused by：Hahn Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteE. coli total protein for SDS PAGEused by：Hah

36、n Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteComassie Blue Protein gel stainused by：Hahn Lab，The Fred Hutchinson Cancer Research Center and Howard Hughes Medical InstituteCoomassie & Silver Staining of Polyacrylamide Gelsused by：Lab of Dr. Mark Barton Frank, Ok

37、lahoma Medical Research Foundation2-D Polyacrylamide Gel Electrophoresisused by：Molecular Profiling Initiative, National Cancer Institute(NCI)Silver Staining of SDS-PAGE Gelsused by：The Minion Lab, College of Veterinary Medicine at Iowa State UniversitySDS-PAGE Gelsused by：The Minion Lab, College of

38、 Veterinary Medicine at Iowa State UniversityCuCl2 Staining of SDS-PAGE Gelsused by：Cepko/Tabin Lab, Harvard UniversityElectroelution of Proteins From SDS-PAGE Gelsused by：Drs. C Cepko and C Tabin, Harvard UniversitySilver Stain of Protein Gelsused by：Dr. DE Koshland, Carnegie Institution of Washing

39、tonSDS-PAGE （BIO-RADused by：Dr. DE Koshland, Carnegie Institution of WashingtonSDS-PAGEused by：Michael Koelles Lab,Department of Molecular Biophysics and BiochemistryYale University9、质谱分析Polycrystalline thin films质谱样品准备used by：Prowl Lab,Rockefeller UniversitySeeded films (Vorm-Roepstorff)used by：Pro

40、wl Lab,Rockefeller UniversityDried dropletused by：Prowl Lab,Rockefeller UniversityIn-gel Tryptic Digest for Protein ID by Mass Spectrometryused by：Mitshison Lab, Department of Systems Biology, Harvard Medical School10、crystallization Polycrystalline thin filmsused by：PROWL, Rockefeller UniversitySlo

41、w crystallizationused by：PROWL, Rockefeller UniversityCrystallization of Kinesin Family Motor Proteins驱动蛋白家族的结晶条件和晶体参数used by：Liz Greene and Steve HenikoffCrystallization Trialsused by：Liz Greene and Steve Henikoff11、in vitro protein synthesis Protein Syntheses in Cell Free Systemsused by：The Labora

42、tory of William Heidcamp at Gustavus Adolphus University12、蛋白纯化/层析 6xHis-tagged protein purification using Qiagen Ni-NTA Column under Native Conditionsused by：Sugden lab,McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical SchoolPurification of dnEBNA-1/Soft from E. coli B

43、L21 LysSused by：Sugden lab,McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical SchoolPurification of GST fusion proteins in E.coli GST融合蛋白纯化（方法四）筛选表达株used by：Sugden lab,McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical SchoolPurification of GST fusion proteins in E.coli GST融合蛋白纯化，方法三，纯化小量蛋白used by：Sugden lab,McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical SchoolPurification of G