Protein Analysis and Purification [electronic resource] : Benchtop Techniques / by Ian M. Rosenberg
- 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 it's a new technique or a task you've 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.
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