Protein Analysis and Purification

1 An Overview of This Manual.- Kits, Cores, and Computers.- How to Use This Book.- Basic Laboratory Equipment.- Laboratory Automation.- Beyond Protein Analysis and Purification.- 2 Protein Structure.- A. The Amino Acids.- B. The Four Levels of Protein Structure.- Primary Structure.- Secondary Structure.- Tertiary Structure.- Quaternary Structure.- C. Chemical Characteristics of Proteins.- Hydrophobicity.- Consensus Sequences.- Proteomics.- 3 Tracking the Target Protein.- A. Labeling Cells and Proteins.- Metabolic Labeling Cells in Culture.- Protocol 3.1 Metabolic Labeling Adherent Cells.- Protocol 3.2 Metabolic Labeling Cells Growing in Suspension.- Protocol 3.3 Pulse-Chase Labeling.- Labeling Proteins Present at the Plasma Membrane.- Protocol 3.4 Lactoperoxidase Labeling Cell Surface Proteins.- Protocol 3.5 Labeling Surface Proteins with IODO-GEN®.- Protocol 3.6 Non-radioactive Biotinylation of Cell Surface Proteins.- Protocol 3.7 Domain-Selective Biotinylation and Streptavidin-Agarose Precipitation.- Protocol 3.8 Labeling Isolated Proteins with Chloramine T.- B. Lysis—Preparation of the Cell Free Extract.- Lysis Buffers.- Protocol 3.9 Lysis of Cells in Suspension (Continuation of Protocol 3.2).- Protocol 3.10 Lysis of Adherent Cells (Continuation of Protocol 3.1).- C. Principles of Immunoprecipitation.- Antibodies as Detection Tools.- Polyclonal Antibodies.- Monoclonal Antibodies.- Antibody Based Analytical Techniques: Western Blotting and Immunoprecipitation.- Protocol 3.11 Immunoprecipitation.- Protein Interaction Analysis.- Protocol 3.12 Sequential Immunoprecipitation: Dissociation and Reimmunoprecipitation of Immune Complexes.- Protocol 3.13 Eliminating Interfering Immunoglobulin Bands During IP-Western Detection: Analysis of the Immunoprecipitate under Non-Reducing Conditions.- Protocol 3.14 Nondenaturing Immunoprecipitation.- D. Additional Methods to Identify Associated Proteins.- Sucrose Gradients.- Protocol 3.15 Preparation of Sucrose Gradients.- Fractionating a Sucrose Gradient.- Chemical Cross-Linking.- General Considerations for Cross-Linking.- Protocol 3.16 Cross-Linking Proteins Added to Cells: Analysis of Receptor-Ligand Interaction.- Protocol 3.17 Cross-Linking Proteins in Solution.- Protocol 3.18 Cross-Linking Extraneously Added Ligand to Cells.- Protocol 3.19 Cross-Linking Proteins in Solution Using the Homobifunctional Reagent Dithiobis (succinimidyl propionate) (DSP).- Analysis of Protein-Protein Interactions.- Mapping Protein-Protein Contact Sites.- Yeast Two-Hybrid Systems.- Analyzing Protein Interactions by Fluorescence Resonance Energy Transfer (FRET).- 4 Electrophoretic Techniques.- to Polyacrylamide Gel Electrophoresis (PAGE).- A. Preparation of SDS-Polyacrylamide Gels.- Protocol 4.1 Assembling the Plates.- Choosing the Acrylamide Concentration.- Protocol 4.2 Casting the Separating Gel.- Protocol 4.3 Casting the Stacking Gel.- Protocol 4.4 Gradient Gels.- Protocol 4.5 Sample Preparation.- Protocol 4.6 Running the Gel: Attaching the Gel Cassette to the Apparatus and Loading the Samples.- Protocol 4.7 Drying the Gel.- Protocol 4.8 Separation of Low Molecular Weight Proteins by Tricine-SDS-PAGE (TSDS-PAGE).- Safety Considerations.- B. 2-Dimensional (2-D) Gel Systems.- Isoelectric Focusing.- Protocol 4.9 Preparation of the Sample for Isoelectric Focusing.- Single-Step Extraction/Solubilization Buffer.- Protocol 4.10 Preparation and Running of Isoelectric Focusing Tube Gels.- Protocol 4.11 Equilibration of the First-Dimension Gel or Strip.- Flaws with 2-D Analysis.- Protocol 4.12 Measuring the pH of the Gel Slices.- Protocol 4.13 Nonequilibrium pH Gradient Electrophoresis (NEPHGE).- Protocol 4.14 2-D Gels—The Second Dimension: SDS-PAGE.- Fluorescence Two-Dimensional Difference Gel Electrophoresis.- (2-D DIGE).- Protocol 4.15 Labeling Proteins with Cyanine Dyes (Cy3 and Cy5).- 2-D PAGE Databases.- Protocol 4.16 Nonreducing-Reducing 2-D Gels.- C. Detection of Protein Bands in Polyacrylamide Gels.- Protocol 4.17 Staining and Destaining the Gel with Coomassie Blue.- Protocol 4.18 Coomassie Staining Using GelCode® Blue.- Protocol 4.19 Staining Gels with SYPRO® Ruby.- Viewing and Imaging a SYPRO Ruby-Stained 1-D or 2-D Gel.- Protocol 4.20 Silver Staining.- Protocol 4.21 Reversible Negative Staining of Proteins in Gels with Imidazole and Zinc Salts.- Protocol 4.22 Molecular Weight Determination by SDS-PAGE.- D. Recovery of Proteins from the Gel.- Protocol 4.23 Excising the Protein Band from the Dried Gel.- Protocol 4.24 Extracting the Target Protein from the Dried Gel.- E. Identification of Enzyme Activity in Polyacrylamide Gels.- General Considerations.- Protocol 4.25 Localization of Proteases: Copolymerization of Substrate in the Separating Gel.- Protocol 4.26 Identification of Protease Inhibitors: Reverse Zymography.- Protocol 4.27 Locating the Enzyme Activity: Reacting the Gel with Substrate Solution after Electrophoresis.- PROTOCOL 4.28 Detection of ?-glucuronidase Activity in.- Polyacrylamide Gels.- Identification of DNA Binding Proteins—Gel Shift Assay.- Protocol 4.29 Gel Shifts.- 5 Getting Started with Protein Purification.- A. Making a Cell Free Extract.- Cellular Disruption.- Extraction Buffer Composition.- Protease Inhibitors.- Methods of Cell Disruption.- Clarification of the Extract.- Protocol 5.1 Nuclear Extracts.- Protocol 5.2 Total Lymphocyte Extract.- Protocol 5.3 Subcellular Fractionation.- Subcellular Markers.- B. Protein Quantitation.- The Bradford Method.- Protocol 5.4 Bradford Standard Assay.- Protocol 5.5 Bradford Microassay.- Protocol 5.6 Protein Determination Using Bicinchoninic Acid (BCA).- Compatible Substances for the BCA Protein Assay.- Incompatible Substances.- Protocol 5.7 NanoOrange® Protein Quantitation Assay: A Fluorescence-Based Assay of Proteins in Solution.- C. Manipulating Proteins in Solution.- Stabilization and Storage of Proteins.- Concentrating Proteins from Dilute Solutions.- Protocol 5.8 Recovery of Protein by Ammonium Sulfate Precipitation.- Ultrafiltration.- Lyophilization.- Dialysis.- Protocol 5.9 Preparation of Dialysis Tubing.- Changing the Buffer by Gel Filtration.- D. Precipitation Techniques.- Protocol 5.10 Salting Out with Ammonium Sulfate.- Protocol 5.11 Precipitation with Acetone.- Precipitation with Polyethylene Glycol (PEG).- Protocol 5.12 PEG Precipitation.- Protocol 5.13 Removal of PEG from Precipitated Proteins.- Precipitation by Selective Denaturation.- Protocol 5.14 Recovery of Protein from Dilute Solutions by Methanol Chloroform Precipitation.- Protocol 5.15 Recovery of Protein by Trichloroacetic Acid (TCA) Precipitation.- Protocol 5.16 Concentration of Proteins by Acetone Precipitation.- What to Do When All Activity Is Lost.- 6 Membrane Proteins.- A. Peripheral Membrane Proteins.- Protocol 6.1 Alkali Extraction.- Protocol 6.2 High pH Membrane Fractionation.- B. Integral Membrane Proteins.- Organic Alcohol Extraction of Peripheral Membrane Proteins.- Protocol 6.3 Butanol Extraction.- Protocol 6.4 Single-Phase Butanol Extraction.- C. Detergents.- Properties of Detergents.- Critical Micelle Concentration (CMC).- Micelle Molecular Weight.- Hydrophile-Lipophile Balance (HLB).- Classification of Detergents.- Ionic Detergents.- Nonionic Detergents.- Bile Salts.- Detergent Solubilization.- Choosing a Detergent.- Choice of Initial Conditions.- Protocol 6.5 Differential Detergent Solubilization.- Protocol 6.6 Solubilization Trial.- Protein-to-Detergent Ratio.- Detergent Removal.- Removal of Ionic Detergents.- Removal of Nonionic Detergents.- Extracti-Gel® D.- 7 Transfer and Detection of Proteins on.- Membrane Supports.- A. Transfer of Proteins to Membrane Supports.- Protocol 7.1 Transfer of Proteins to Nitrocellulose or Polyvinylidene Difluoride.- Troubleshooting Western Blots.- Protocol 7.2 Enhanced Capture of Small Histidine Containing Polypeptides on Membranes in the Presence of ZnCl2.- Protocol 7.3 Dot Blots.- Protocol 7.4 Thin-Layer Chromatography Blotting.- B. Staining the Blot.- Protocol 7.5 Total Protein Staining with India Ink.- Protocol 7.6 Reversible Staining with Ponceau S.- Protocol 7.7 Irreversible, Rapid Staining with Coomassie Brilliant Blue.- Protocol 7.8 Staining Immobilized Glycoproteins by Periodic Acid/Schiff (PAS).- C. Recovery of Proteins from the Blot.- Protocol 7.9 Recovery of Proteins Using an Organic Solvent System.- Protocol 7.10 Recovery of Proteins from the Blot Using a Detergent-Based Solvent System.- Protocol 7.11 Blocking the Blot.- Protocol 7.12 Exposing the Blot to Primary Antibody.- Ligand Blotting.- Southwestern Blotting.- Far Western Blotting.- Protocol 7.13 Ligand Binding.- Protocol 7.14 Lectin Blotting.- Protocol 7.15 Bacterial Protein Overlay Analysis.- D. Detection of the Target Protein.- Protein A.- Second Antibody Conjugate.- Biotin Avidin System.- Protocol 7.16 Biotinylation of Proteins.- Protocol 7.17 Purifying and Biotinylating Antibodies from Immunoblots.- Enzymatic Detection Methods.- Horseradish Peroxidase.- Protocol 7.18 Colorimetric Detection with Diaminobenzidine, 3,3’,4,4’-tetraaminobiphenyl) (DAB).- Protocol 7.19 Colorimetric Detection Using Alkaline Phosphatase.- Protocol 7.20 Enhanced Chemiluminescence.- Protocol 7.21 Stripping and Reprobing the Blot.- Detection of Radiolabeled Proteins.- Protocol 7.22 Direct Autoradiography.- Removing Unwanted Background Signal from X-ray Film.- Protocol 7.23 Phosphorimaging.- 8 Identification of the Target Protein.- A. Peptide Mapping.- Protocol 8.1 Thermal Denaturation.- Preparing the Target Protein for Digestion.- B. Enzymatic Cleavage of Proteins.- Protocol 8.2 Peptide Mapping by Proteolysis and Analysis by Electrophoresis.- Cleavage of Proteins Transferred to PVDF or NC Membranes.- Protocol 8.3 Cleavage of Proteins Immobilized on the Membrane.- Protocol 8.4 Tryptic Cleavage of Protein Eluted from PVDF Membrane.- C. Chemical Cleavage.- Protocol 8.5 Cyanogen Bromide Cleavage of Proteins on PVDF Membrane.- Protocol 8.6 N-Chlorosuccinimide (NCS) Mapping.- Protocol 8.7 Hydroxylamine Cleavage of Proteins in Polyacrylamide Gels.- Protocol 8.8 Formic Acid Cleavage.- Protocol 8.9 Chemical Cleavage at Cysteine Residues with DTNB.- D. Microsequencing from PVDF Membranes.- When, and In What Form Do You Submit the Target Protein to the Protein Sequencing Core?.- Protocol 8.10 Transferring Spots from 2-D Gels to PVDF Membranes.- Sequencing Glycopeptides.- Protocol 8.11 Protein Hydrolysis: Total Amino Acid Composition of the Target Protein.- Identifying Proteins Using Mass Spectrometry.- Preparation of Proteins for MS Analysis.- Partial Proteolysis.- Protocol 8.12 In-gel Tryptic Digestion.- Protocol 8.13 Extraction of Peptides from Gel Pieces Containing Integral Membrane Proteins.- Elution of Target Protein from SDS-PAGE.- Protein Transfer to a Membrane.- Considerations.- MS Basics.- Electrospray and Tandem Mass Spectrometry.- MALDI and Peptide-Mass Mapping.- Post-Source Decay (PSD) MALDI-MS.- Protein Identification by MS.- Peptide Mass Fingerprint Analysis.- Peptide Fragmentation.- Peptide Ladder Sequencing.- Peptide Sequence Tag.- Identification of a Gene Product.- Posttranslational Modifications and MS.- Caveats When Using MS.- Protein Database Searches.- Bioinformatics.- The Rise of Biological Databases.- Search Engines.- Databases.- Identifying a Target Protein.- Gene Analysis Tools.- Subcellular Localization.- Protein Domain Families.- Structural Classification of Proteins.- Genomic Organization.- Additional Useful Sites on the Internet.- PCR Primer Design Programs.- Microarrays and Data Mining—the Challenge of Data Analysis.- 9 Identifying and Analyzing Posttranslational.- Modifications.- A. Glycosylation.- Protocol 9.1 Chemical Deglycosylation Using Trifluoromethanesulfonic Acid (TFMS).- N-Glycosylation.- Protocol 9.2 Removal of the Oligosaccharide from the Glycoprotein with N-Glycanase.- Protocol 9.3 N-glycosidase F (GPase F) Treatment of Glycoproteins in Immunoprecipitates.- Protocol 9.4 Tunicamycin.- O-Glycosylation.- Protocol 9.5 Identification of O-Glycosylated Amino Acids by Alkaline 13-Elimination.- Protocol 9.6 ?-Elimination of O-Glycans from Glycoproteins Immobilized on Blots.- Protocol 9.7 O-Glycosidase.- Protocol 9.8 O-Glycanase.- Combined Use of N-Glycanase and O-Glycanase.- Protocol 9.9 Endoglycosidase H.- Neuraminidase (NA).- Protocol 9.10 Desialylation with Clostridium perfringens Neuraminidase.- Protocol 9.11 Desialylation with Arthrobacter ureafaciens Neuraminidase.- Lectins as Tools for Carbohydrate Analysis.- Proteoglycans.- Protocol 9.12 Is the Target Protein a Proteoglycan?.- B. Phosphorylation.- Protocol 9.13 Metabolic Labeling of Cells with [32P]orthophosphate.- Protocol 9.14 Can the Target Protein Be Phosphorylated?.- Protocol 9.15 Determination of the Type of Phosphorylated Amino Acid-Immunoblotting with Anti-Phosphoamino Acid Antibodies.- Protocol 9.16 Phosphorylation of Membrane Proteins with [?-32P]GTP.- Enzymatic Dephosphorylation.- Protocol 9.17 Potato Acid Phosphatase.- Protocol 9.18 Alkaline Phosphatase.- Protocol 9.19 Immune Complex Kinase.- Protocol 9.20 Renaturation of Immobilized Kinases on PVDF Membranes.- Protocol 9.21 Phosphorylation of Substrates in SDS-Gels.- Phosphopepetide and Phosphoamino Acid Analysis.- Protocol 9.22 One-Dimensional Phosphopeptide Mapping.- Two-Dimensional Phosphopeptide Mapping.- Protocol 9.23 Isolation of Phospho-Proteins from SDS Gels: Preparation for Phosphopeptide Mapping.- Protocol 9.24 Tryptic Digestion of Isolated Phosphoproteins.- Protocol 9.25 Applying the Sample to the TLC Plate and Electrophoresis in the First Dimension.- Protocol 9.26 Second Dimension: Thin-Layer Chromatography.- Protocol 9.27 Isolation of Individual Phosphopeptides from TLC Plates.- Protocol 9.28 Phosphoamino Acid Analysis.- Protocol 9.29 Phosphoamino Acid Analysis of Phosphoproteins Isolated from PVDF Membranes.- Protocol 9.30 Identification of Phosphohistidine Residues Following Heat Treatment.- Protocol 9.31 Treatment with Diethyl Pyrocarbonate.- Protocol 9.32 Treatment of Phosphorylated Membranes with HC1 and NaOH.- Protocol 9.33 Sulfation.- C. Lipid Modification of Proteins.- Palmitoylation and N-Myristoylation of Proteins.- Analysis of Bound Fatty Acids.- Protocol 9.34 Identification of Palmitoylated and Myristoylated Proteins.- Isoprenylation.- Protocol 9.35 Metabolic Labeling with [3H]Mevalonic Acid Derivatives.- Protocol 9.36 Enzymatic Prenylation of Recombinant Proteins.- Glypiation.- Is the Target Protein Glycosyl Phosphatidylinositol Anchored?.- Use of Triton X-114.- Protocol 9.37 Preparation of Triton X-114.- Protocol 9.38 Fractionation of Integral Membrane Proteins with Triton X-114.- Protocol 9.39 Digestion with Phosphatidylinositol Specific Phospholipase C (PI-PLC).- Metabolic Labeling with Precursors of the GPI Structure.- Use of Anti-CRD.- D. Selected Modifications.- Transamidation.- Acetylation.- Methylation.- Hydroxylation of Proline and Lysine.- Degradation.- Ubiquitination.- Proteolytic Processing.- 10 Chromatography.- A. Important Terminology Used in Chromatography.- B. Gel Filtration Chromatography.- Choice of Buffer.- Choice of Column Size.- Protocol 10.1 Preparation of the Gel: Hydrating and Degassing.- Protocol 10.2 Packing the Column.- Flow Rate.- Hydrostatic Pressure.- Sample Application.- Protocol 10.3 Loading Sample onto a Drained Bed.- Loading Sample Under the Eluent.- Making Sure the Column Does Not Run Dry.- Molecular Weight Determination.- Spin Columns Used in Gel Filtration.- Protocol 10.4 Spin Columns.- Protocol 10.5 Testing Fractions to Locate Protein: Bradford Spot Test.- C. Introduction to HPLC.- Packing Materials.- Column Designs.- Column Guards.- Detectors.- Choosing the Right Conditions—Some Helpful Tips.- HPLC—Size Exclusion.- D. Ion Exchange Chromatography: Separation on the Basis of Charge.- Simplified Theory of Ion Exchange.- Functional Groups on Exchange Columns.- Choice of Exchanger Matrix.- Preparation of the Exchanger.- Choice of Buffer.- Batch Adsorption.- Protocol 10.6 Selecting the Starting pH.- Protocol 10.7 Packing an Ion Exchange Column.- Experimental Tips.- Elution-Step or Linear Gradient?.- Protocol 10.8 Regeneration of Sephadex Ion Exchangers.- Protocol 10.9 Regeneration of Sepharose Ion Exchangers.- Protocol 10.10 Chromatofocusing.- Removing the Polybuffer.- HPLC-Ion Exchange Chromatography.- Membrane Adsorbers.- Perfusion Chromatography.- E. Hydrophobic Interaction Chromatography (HIC).- Simplified Theory of HIC.- Protocol 10.11 Protein Fractionation by HIC.- Protocol 10.12 Solid Phase Extraction Cartridges.- Reversed Phase HPLC.- Reversed Phase HPLC for the Isolation of Peptides.- Multidimensional Liquid Chromatography.- Affinity Chromatography.- Immunoaffinity Purification.- Protocol 10.13 Direct Antibody Coupling to Protein A Beads.- Protocol 10.14 Indirect Antibody Coupling to Protein A Beads.- Protocol 10.15 Preparation of Affinity Columns.- Flow Rate.- Binding Antigens to Immunoaffinity Matrices.- Nonspecific Interactions.- Protocol 10.16 Blocking the Affinity Matrix.- Elution of Antigens from Immunoaffinity Matrices.- Protocol 10.17 Eluting the Antigen.- Ligand Affinity Chromatography.- Protocol 10.18 Immobilization of Proteins to N-Hydroxysuccinimide Ester Derivatives of Agarose.- Toluene Sulfonyl Chloride (Tosyl Chloride).- Pseudo-Affinity Adsorbents.- Lectin Affinity Chromatography.- Protocol 10.19 Glycoprotein Purification Using Wheat Germ Agglutinin (WGA).- Protocol 10.20 Immobilized Metal Chelate Chromatography (IMAC).- Protocol 10.21 Purifying a Histidine Tagged Recombinant Fusion Protein.- Protocol 10.22 Hydroxylapatite Chromatography.- 11 Recombinant Protein Techniques.- Recombinant Protein for Antibody Production.- Protein for Biochemical or Cell Biological Studies.- A. In vitro Transcription and Translation.- Protocol 11.1 Preparation of the DNA Template.- Protocol 11.2 In vitro Transcription—Preparation of the mRNA.- Protocol 11.3 Guanylyltransferase Catalyzed Addition of a G(5’)ppp(5’)G Cap to mRNA.- Protocol 11.4 In vitro Translation: Protein Synthesis.- Protocol 11.5 Cotranslational Processing Using Canine Pancreatic Microsomal Membranes.- Protocol 11.6 Translocated Products Are Resistant to Protease Digestion.- Protocol 11.7 Was the Translational Product Glycosylated? Endoglycosidase H (Endo H) Analysis.- Protein Transduction: A Method for Introducing Exogenous Proteins into Cells.- B. Recombinant Gene Products in E. coli: Expression, Identification and Characterization.- Expression and Purification of lacZ and trpE Fusion Proteins.- Protocol 11.8 lacZ Induction.- Protocol 11.9 Induction of the trpE Fusion Protein.- Protocol 11.10 Preparation of the Protein Extract.- Protocol 11.11 Solubilization of the Fusion Protein.- Protocol 11.12 Purification of Eukaryotic Proteins from Inclusion Bodies in E. coli.- Glutathione-S-Transferase (GST) Fusion Proteins.- Protocol 11.13 Production and Analysis of GST Fusion Protein Transformants (Small Scale).- Protocol 11.14 Purification of GST Fusion Proteins.- Protocol 11.15 Removing the GST from the Fusion Protein.- Protocol 11.16 His-Tag Purification System.- Maltose Binding Protein (MBP) Fusion Proteins.- Staphylococcal Protein A and ZZ.- Green Fluorescent Protein (GFP).- D. Expression of Foreign Proteins in Eukaryotic Cells.- Expression and Isolation of Recombinant Proteins from Yeast.- Protocol 11.17 Preparation of Protein Extracts from Yeast.- Expression of Proteins in Insect Cells Using Baculoviral Vectors.- Expression of Foreign Proteins in Mammalian Cells.- Transfection: Expression of Recombinant Proteins in Eukaryotic Systems.- Protocol 11.18 Transfection of DNA into Eukaryotic Cells with Calcium Phosphate.- Protocol 11.19 Glycerol Shock.- Protocol 11.20 Transfection Using DEAE-Dextran.- Protocol 11.21 Stable Transfections.- Protocol 11.22 Picking Stable Colonies.- Appendices.- A. Safety Considerations.- First Aid: Emergency Procedures.- B. Antibody Preparation.- Production of Polyclonal Antisera in Rabbits.- Purification of Antibody Using Protein A Affinity Columns.- Numbering Mice.- Molarity.- Choosing and Preparing Buffers.- Common Laboratory Solutions.- Extinction Coefficients.- D. Nucleic Acids.- Spectrophotometric Conversions.- DNA/Protein Conversions.- Oligonucleotide Concentrations.- RNA Precipitation.- E. Modifications and Motifs.- Nomenclature.- Protein Modification Sequences.- Protein Kinase Recognition Sequence Motifs.- Subcellular Localization Motifs.- Protein Databases.- F. Centrifugation.- Nomogram.- General Purpose Centrifuge Rotors.- Ultracentrifuge Rotors.- G. Proteases and Proteolytic Enzyme Inhibitors.- Commonly Used Proteases.- Protease Inhibitors.- H. Radioactivity.- Manual and Machine Film Processing.- L Tissue Culture.- Transwell Permeable Supports.- J. Miscellaneous.- Unit Prefixes.- The Greek Alphabet.- Abbreviations.- HPLC Pump Pressure Conversion.- Dipeptide Masses.- Mass Differences Considered in Molecular Weight Analysis of Proteins.- K. List of Suppliers, Vendors, Manufacturers.