Description
BIRKHAUSER BOSTON Protein Analysis And Purification by Ian M. Rosenberg
How one goes about analyzing proteins is a constantly evolving field that is no longer solely the domain of the protein biochemist. Investi gators from diverse disciplines find themselves with the unanticipated task of identifying and analyzing a protein and studying its physical properties and biochemical interactions. In most cases, the ultimate goal remains understanding the role(s) that the target protein is playing in cellular physiology. It was my intention that this manual would make the initial steps in the discovery process less time consuming and less intimidating. This book is not meant to be read from cover to cover. The expanded Table of Contents and the index should help locate what you are seeking. My aim was to provide practically oriented information that will assist the experimentalist in benchtop problem solving. The appendices are filled with diverse information gleaned from catalogs, handbooks, and manuals that are presented in a distilled fashion designed to save trips to the library and calls to technical service representatives. The user is encouraged to expand on the tables and charts to fit individual experimental situations. This second edition pays homage to the computer explosion and the various genome projects that have revolutionized how benchtop scientific research is performed. Bioinformatics and In silica science are here to stay. However, the second edition still includes recipes for preparing buffers and methods for lysing cells. 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 (R).- 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 (R) Blue.- Protocol 4.19 Staining Gels with SYPRO (R) 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 (R) 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 (R) 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 Radiolabele dProteins.- Protocol 7.22 Direct Auty- 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.