Guide to Protein Purification, Second Edition

✍ Scribed by Richard R Burgess, Murray P. Deutscher

• Publisher: Elsevier, Academic Press
• Year: 2009
• Tongue: English
• Leaves: 854
• Series: Methods in Enzymology 463
• Edition: 2
• Category: Library

✦ Synopsis

The 2eof this classic Guide to Protein Purification provides a complete update to existing methods in the field, reflecting the enormous advances made in the last two decades. In particular, proteomics, mass spectrometry, and DNA technology have revolutionized the field since the first edition's publication but through all of the advancements, the purification of proteins is still an indispensable first step in understanding their function. This volume examines the most reliable, robust methods for researchers in biochemistry, molecular and cell biology, genetics, pharmacology, biotechnology and sets a standard for best practices in the field. It relates how these traditional and new cutting-edge methods connect to the explosive advancements in the field. This "Guide to" gives imminently practical advice in order to avoid costly mistakes in choosing a method and brings in perspective from the premier researchers while it presents a comprehensive overview of the field today. Gathers top global authors from industry, medicine, and research fields across a wide variety of disciplines including biochemistry, genetics, oncology, pharmacology, dermatology and immunologyAssembles chapters on both common and less-common relevant techniquesProvides robust methods as well as an analysis of the advancements in the field that, for an individual investigator, can be a demanding and time-consuming process

✦ Table of Contents

Methods in Enzymology
......Page 2
Insert from: "51.pdf"......Page 0
Why Purify Enzymes?
......Page 55
Acknowledgment
......Page 58
Strategies and Considerations for Protein Purifications......Page 59
Proteases at room temperature......Page 60
Glycosylation......Page 61
Ubiquitination and sumoylation......Page 63
Buffers......Page 64
Chromatography......Page 65
EDTA binding to anion-exchange columns
......Page 66
Method......Page 67
Sedimentation velocity......Page 68
References......Page 69
Use of Bioinformatics in Planning a Protein Purification......Page 70
What You Can Learn from an Amino Acid Sequence......Page 71
Molar extinction coefficient......Page 72
Multisubunit features; homomultimers, heteromultimers?......Page 75
Protocol......Page 76
Preparing a Purification Summary Table......Page 78
The Value of an SDS-Polyacrylamide Gel Analysis on Main Protein Fractions......Page 81
Setting Up a Laboratory......Page 84
Chemicals......Page 85
Small equipment and accessories......Page 86
Introduction......Page 90
Theory......Page 91
Buffer Selection......Page 92
Volatile Buffers......Page 96
Broad-Range Buffers......Page 97
Measurement of Enzyme Activity......Page 104
Introduction......Page 105
Discussion......Page 118
Quantitation of Protein......Page 119
Ultraviolet absorbance at 280 nm (Range: 20-3000 mug)......Page 126
Dye-Based Protein Assays......Page 129
Coomassie Blue (Bradford) Protein Assay (Range: 1-50 mug)......Page 131
Comments......Page 132
Gel-filtration (size-exclusion) chromatography......Page 133
Problems and pitfalls......Page 134
Method......Page 135
Field-flow fractionation......Page 136
Problems and pitfalls......Page 137
Scattering Methods......Page 138
Electron microscopy......Page 139
References......Page 140
Concentration of Proteins and Removal of Solutes......Page 142
Electrophoresis......Page 148
Dialysis......Page 149
Precipitation......Page 161
Crystallization......Page 163
Maintaining Protein Stability......Page 166
General Handling Procedures......Page 167
Selecting an Appropriate Method for Expressing a Recombinant Protein
......Page 173
Introduction......Page 174
Escherichia coli......Page 175
E. coli: Disulfide bond formation......Page 176
Pichia pastoris......Page 177
Mammalian Cells......Page 180
Recombinant Protein Applications......Page 185
Bacterial Systems for Production of Heterologous Proteins
......Page 190
Heterologous Protein Production Using Escherichia coli
......Page 191
Planning a Bacterial Expression Project......Page 192
Target Analysis......Page 193
Cloning......Page 194
Preparation of T4 DNA Polymerase-Treated DNA Fragments......Page 196
Expression in the E. coli Cytoplasm......Page 197
Analysis of Heterologous Protein Expression in E. coli......Page 198
Analysis of protein solubility......Page 199
Small-Scale Expression Cultures in Autoinduction Media Protocol
......Page 201
Small-Scale Osmotic Shock Protocol......Page 203
Alternative Bacterial Systems for Heterologous Protein Production
......Page 205
Acknowledgment
......Page 207
Expression in the Yeast Pichia pastoris
......Page 210
Introduction......Page 211
Genetic Strain Construction......Page 213
Gene Preparation and Vector Selection......Page 215
Baculovirus-Insect Cell Expression Systems......Page 231
Introduction......Page 232
A Brief Overview of Baculovirus Biology and Molecular Biology
......Page 233
Baculovirus Expression Vectors......Page 235
Baculovirus Expression Vector Technology-The Early Years
......Page 236
Baculovirus Transfer Plasmid Modifications......Page 238
Parental Baculovirus Genome Modifications......Page 240
The Other Half of the Baculovirus-Insect Cell System......Page 250
A New Generation of Insect Cell Hosts for Baculovirus Expression Vectors
......Page 252
References......Page 258
Recombinant Protein Production by Transient Gene Transfer into Mammalian Cells
......Page 263
HEK293 and CHO Cell Lines Commonly Used in TGE Approaches
......Page 264
Expression Vectors for HEK293 and CHO Cells......Page 266
Transfection Methods......Page 268
Large-scale transient transfection in the Wave Bioreactor with polyethylenimine as transfer reagent
......Page 272
Acknowledgments......Page 274
References......Page 275
Tagging for Protein Expression
......Page 279
Introduction......Page 280
Affinity and/or solubility?
......Page 281
Which tag(s) to use?
......Page 283
Protein Affinity Tags......Page 285
Solubility Tags......Page 289
References......Page 294
Refolding Solubilized Inclusion Body Proteins......Page 299
Introduction......Page 300
General Procedures......Page 302
General Protocol......Page 303
Overexpression of recombinant proteins in E. coli......Page 304
Solubilizing IBs......Page 305
Refolding......Page 307
Characterization......Page 310
Variables in refolding......Page 311
A practical, inexpensive rational approach......Page 314
Other Refolding Procedures......Page 315
Strategies to Increase Proportion of Soluble Protein......Page 317
References......Page 319
Advances in Preparation of Biological Extracts for Protein Purification
......Page 323
Introduction......Page 324
Chemical and Enzymatic Cell Disruption......Page 325
Mechanical Cell Disruption......Page 328
Buffer composition......Page 331
Microscale protocols for E. coli cell lysis......Page 334
Freeze-thaw plus enzymatic lysis......Page 336
High-pressure homogenization......Page 338
References......Page 339
Isolation of Subcellular Organelles and Structures......Page 342
Introduction......Page 357
Introduction......Page 361
Introduction......Page 362
Protein Precipitation Techniques......Page 366
Introduction......Page 367
Basic procedure......Page 369
Comments/problems/solutions......Page 372
Different modes of use of PEI......Page 373
Doing an PEI precipitation test......Page 375
Other Methods......Page 376
Affi-Gel Blue for Nucleic Acid Removal and Early Enrichment of Nucleotide Binding Proteins
......Page 378
Reference
......Page 380
Ion-Exchange Chromatography......Page 381
Elution Conditions......Page 393
Operation of Ion-Exchange Columns......Page 395
Example: Separation of Complex Protein Mixture......Page 398
Example: High-Resolution Separation with a Monolithic Column
......Page 399
Gel Filtration......Page 404
Matrices......Page 407
Preliminary screening......Page 411
Protein Chromatography on Hydroxyapatite Columns......Page 417
Introduction......Page 418
Mechanisms......Page 419
Chemical Characteristics......Page 422
Metal adsorption......Page 423
Packing Laboratory-Scale Columns......Page 427
Applications......Page 431
Theory and Use of Hydrophobic Interaction Chromatography in Protein Purification Applications
......Page 435
Theory......Page 436
Latest Technology in HIC Adsorbents......Page 438
Choice of adsorbent......Page 439
Adsorbent preparation......Page 440
Stepwise (isocratic) elution......Page 442
References......Page 443
Affinity Chromatography: General Methods......Page 445
Selection of Ligands......Page 451
Purification Method......Page 461
Immobilized-Metal Affinity Chromatography (IMAC): A Review......Page 467
Overview on IMAC Ligands and Immobilized Ions......Page 468
IMAC Applications......Page 472
Detection and immobilization......Page 473
General considerations of protein purification by IMAC
......Page 479
Purification of His-tagged proteins under native conditions......Page 493
References......Page 496
Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography
......Page 502
Introduction......Page 503
Polyol-Responsive Monoclonal Antibodies......Page 504
Purification of proteins using cross-reacting PR-mAbs
......Page 517
References......Page 520
One-Dimensional Gel Electrophoresis
......Page 522
Polyacrylamide Gels......Page 525
Principle of Method......Page 526
Procedure......Page 527
Casting gels......Page 528
Sample preparation......Page 530
Molecular Weight Determination......Page 536
Isoelectric Focusing and Two-Dimensional Gel Electrophoresis......Page 539
Introduction......Page 540
Materials......Page 551
Protein Gel Staining Methods: An Introduction and Overview
......Page 565
Introduction......Page 566
Instrumentation: Detection and Documentation
......Page 569
SYPRO Ruby protein gel stain and other Ruthenium-based formulations
......Page 577
Epicocconone: Deep Purple, Lighting Fast protein gel stains......Page 578
Krypton protein gel stains......Page 579
Phosphoprotein Detection......Page 580
Fluorescent general glycoprotein stains
......Page 582
Azide-alkyne ``click chemistry´´ reagents
......Page 583
Elution of Proteins from Gels......Page 588
Elution of Proteins from Gels by Diffusion.....Page 589
SDS removal and concentration
......Page 591
Electrophoretic Elution......Page 593
Conclusion......Page 594
Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection
......Page 596
Assay methods......Page 601
Fluorescence detection......Page 604
The Chemiluminescence Signal......Page 606
Blocking buffer......Page 609
Detection method......Page 610
No signal......Page 611
High background......Page 612
Ghost/hollow bands......Page 613
Western blotting protocol using chemiluminescent substrates
......Page 616
Protocol......Page 618
Optimizing membrane blocking......Page 619
Isotopic Labeling for Structural Studies......Page 620
Optimizing the detection method......Page 621
Detergents: An Overview
......Page 623
Detergent Structure......Page 624
Properties of Detergents in Solution......Page 625
Phase diagrams and critical micellization concentration
......Page 629
Effects of temperature: Detergent solubility, krafft point, cloud point, and phase separation......Page 631
Exploiting the Physicochemical Parameters of Detergents for Membrane Protein Purification
......Page 632
Choosing the Right Detergent......Page 633
Optical spectroscopy......Page 634
Protein crystallization......Page 635
Acknowledgments......Page 636
Purification of Membrane Proteins......Page 638
Purification of Membrane Proteins......Page 644
Lectin-affinity chromatography
......Page 645
Antibody-affinity chromatography......Page 646
Detergent Removal and Detergent Exchange......Page 647
Purification of Recombinant G-Protein-Coupled Receptors
......Page 649
Introduction......Page 650
Solubilization: General Considerations......Page 651
Purification: General Considerations......Page 652
Solubilization and Purification of a Recombinant Neurotensin Receptor NTS1
......Page 655
Purification of the NTS1 fusion protein by a neurotensin column
......Page 658
Analysis of Purified NTS1......Page 659
Conclusions......Page 660
Cell-Free Translation of Integral Membrane Proteins into Unilamelar Liposomes
......Page 664
Introduction......Page 665
Overview of Cell-Free Translation......Page 667
Expression Vectors......Page 668
Gene Cloning......Page 669
Materials and reagents......Page 671
Transformation Reaction......Page 675
Purification of Plasmid DNA......Page 676
Wheat Germ Translation Reaction......Page 679
Materials and reagents......Page 680
Purification by Density Gradient Ultracentrifugation
......Page 682
Characterization of Proteoliposomes......Page 684
Considerations for Scale-Up......Page 686
Conclusions......Page 687
Determination of Protein Purity
......Page 691
Composition-Based and Activity-Based Analyses
......Page 694
Electrophoretic Methods......Page 695
Pitfalls and problems......Page 698
Method......Page 699
Method......Page 701
Determination of Size, Molecular Weight, and Presence of Subunits
......Page 704
Introduction......Page 705
Overview......Page 708
Overview......Page 710
Overview......Page 711
Overview......Page 714
Method......Page 715
Variations......Page 718
Presence of Subunits......Page 732
References......Page 734
Identification and Quantification of Protein Posttranslational Modifications
......Page 737
Introduction......Page 738
Phosphorylation......Page 743
Mass Spectrometry Analysis......Page 756
CID versus ECD versus ETD......Page 759
Quantifying PTMs......Page 762
Future Directions......Page 768
Parallel Methods for Expression and Purification......Page 776
Strategies Based on End-Use......Page 777
Parallel Cloning Strategies for Creating Expression Constructs
......Page 780
Small-Scale Expression Screening to Identify Suitable Constructs
......Page 783
Analytical Testing of Proteins for Selection......Page 787
Large-Scale Parallel Expression......Page 789
Conclusion......Page 792
Techniques to Isolate O2-Sensitive Proteins: [4Fe-4s]-FNR as an Example
......Page 795
Introduction......Page 796
General preparation for achieving and maintaining an oxygen-free environment
......Page 798
Expression of FNR and assembly of [4Fe-4S] clusters
......Page 799
Preparation of cell lysate
......Page 800
Protocols......Page 802
Removal of trace RNAses from purified 4Fe-FNR for in vitro transcription assays
......Page 805
Purification of 57Fe labeled 4Fe-FNR for Mössbauer spectroscopic analysis
......Page 806
Characterization of [4Fe-4S]2+ Cluster Containing FNR
......Page 807
Fe and sulfide analysis2
......Page 808
ICP-MS analysis of the 57Fe content in the 57Fe labeled 4Fe-FNR
......Page 809
Low-temperature EPR
......Page 810
References......Page 811
Introduction......Page 814
Important but Little Known (or Forgotten) Artifacts in Protein Biochemistry
......Page 817
SDS Gel Electrophoresis Sample Preparation......Page 818
Asp-Pro bond cleavage at 100 degC
......Page 819
Keratin contamination in sample or SDS sample buffer
......Page 820
Protein Absorption During Filtration
......Page 822
Cyanate in Urea......Page 823
References......Page 824

✦ Subjects

Биологические дисциплины;Биохимия;Энзимология;