Protein Analysis and Purification : Benchtop Techniques 1 An Overview of this Manual 2 Protein Structure 3 Tracking the Target Protein 4 Electrophoretic Techniques 5 Getting Started with Protein Purification 6 Membrane and Particulate Associated Proteins 7 Transfer and Detection of Proteins on Membrane Supports 8 Peptide Mapping and Microsequencing 9 Modified Proteins and Peptides 10 Chromatograph 11 Recombinant Protein Techniques A. Safety Considerations B. Antibody Preparation Production of Polyclonal Antisera in Rabbits Protocol B.1 Preparation of the Antigen Adjuvant Emulsion Protocol B.2 Intramuscular Immunization (IM) Protocol B.3 Intradermal Immunization Protocol B.4 Subcutaneous Immunization Protocol B.5 Bleeding the Rabbit and Serum Preparation Protocol B.6 Precipitation of IgG with Saturated Ammonium Sulfate Purification of Antibody Using Protein A Affinity Columns Protocol B.7 Purifying Total Ig Numbering Mice C. Solutions Commercial Strengths of Common Laboratory Chemicals Water Molarity Choosing and Preparing Buffers Common Laboratory Solutions Extinction Coefficients D. Nucleic Acids Spectrophotometric Conversions DNA Protein Conversions Oligonucleotide Concentrations Protocol D.1 DNA Quantitation Assay The Codon Dictionary Amino Acid Code Degeneracy Amino Acid and Codon Usage E. Modifications and Motifs Nomenclature Protein Modification Sequences Protein Kinase Recognition Motifs Subcellular Localization Motifs Protein Databases E Centrifugation Nomogram General Purpose Centrifuge Rotors. Ultracentrifuge Rotors G. Proteases and Proteolytic Enzyme Inhibitors Commonly Used Proteases Protocol G.1 Preparation of Defatted BSA Protease Inhibitors H. Radioactivity Manual and Machine Film Processing I. Miscellaneous Unit Prefixes The Greek Alphabet Abbreviations HPLC Pump Conversion Protocol I.1 Siliconizing Glassware J. List of Suppliers Vendors Manufacturers References. This book is designed to be a practical progression of experimental techniques an investigator may follow when embarking on a biochemical project. The protocols may be performed in the order laid out or may be used inde­ pendently. The aim of the book is to assist a wide range of researchers. from the novice to the frustrated veteran in the choice and design of experiments that are to be performed to provide answers to specific questions. The manual describes standard techniques that have been shown to work as well as some newer ones that are beginning to prove important. By following the promi­ nently numbered steps. you can work your way through any protocol. whether its a new technique or a task youve done before for which you need a quick review or updated methodology. This manual will assist the experimentalist in designing properly controlled experiments. There will be no advice for dealing with specific pieces of equip­ ment other than encouragement to read the manual if you can find it. Through­ out all manipulations try to be objective. Be on the lookout for unexpected findings. You will learn the most from unexpected results. and they are often the beginning of the next project. It is never possible to record too much in your lab notebook. Do not get discouraged. Remember things will not always run smoothly.