New Protein Techniques: (3 Methods in Molecular Biology)

Inrecent years therehasbeen atremendousincreaseinour understandingofthe functioningofthe cellat the molecular leveL This has been achieved in the main by the invention and developmentof new methodology, particularlyin that area generally referred to as "genetic engineering. " Al- though this revolution has been taking place in the field of nucleic acids research, the protein chemist has at the same timedevelopedfresh methodologytokeeppace with the re- quirements ofpresent-daymolecularbiology. Today's mo- lecularbiologistscannolongerbecontentwithbeingexperts inoneparticulararea alone. Theyneedtobeequallycompe- tentin the laboratory at handling DNA, RNA,and proteins movingfrom one area toanotherasrequiredby theproblem thatisbeing solved. Althoughmanyof thenewtechniques in molecularbiologyare relativelyeasy tomaster, itisoften difficult for a researcher to obtain all the relevant informa- tionnecessaryforsettingupandsuccessfullyapplyinganew technique. Informationisofcourse availablein the research literature, but this often lacks the depth of description that the new user requires. This requirement for in-depth prac- tical details has become apparent by the considerable de- mand for places on our Molecular BiologyWorkshops held at Hatfield each summer. Volume 1of this series describedpracticalprocedures for a range of protein techniques frequently used by research workers in the field of molecular biology. Because of the limitations on length necessarily inherent in producing any v vi Preface book, one obviouslyhad to be selective in the choiceof titles forVolume1. TheproductionofVolume 3,therefore,allows the development of the theme initiated in Volume 1. This volumecontains afurther selection ofdetailed protocols for arangeofanalyticalandpreparativeproteintechniques,and should be seen as a continuation of Volume 1. Companion Volumes2and4provideprotocols fornucleic acid method- ology. Each methodisdescribedby an authorwhohas regularly used the technique in his or her ownlaboratory.

Table of Contents:
Prevention of Unwanted Proteolysis.- The Bradford Method for Protein Quantitation.- Amino Acid Analysis by Precolumn Derivatization.- Identification of N-Terminal Amino Acids by High-Performance Liquid Chromatography.- Enzymatic Methods for Cleaving Proteins.- Chemical Cleavage of Proteins.- Chemical Modification of Proteins.- The Design, Preparation, and Use of Immunopurification Reagents.- Dye-Ligand Chromatography.- Aminohexyl-Sepharose Affinity Chromatography.- Purification of DNA-Dependent RNA Polymerase from Eubacteria.- Direct Immunoprecipitation of Protein.- Detection of Proteins in Polyacrylamide Gels Using an Ultrasensitive Silver Staining Technique.- Chromatofocusing.- Hybrid Isoelectric Focusing Using Mixed Synthetic-Carrier Ampholyte-Immobilized pH Gradient Gels.- Two-Dimensional Electrophoresis Using Immobilized pH Gradients in the First Dimension.- Two-Dimensional Polyacrylamide Gel Electrophoresis Using Flat-Bed Isoelectric Focusing in the First Dimension.- Preparative Aspects of Immobilized pH Gradients.- Isoelectric Focusing Under Denaturing Conditions.- Computer Analysis of 2-D Electrophoresis Gels.- Two-Dimensional (Crossed) Immunoelectrophoresis.- Peptide Synthesis.- Synthesis of a Series of Analogous Peptides Using T-Bags.- Production of Antisera to Synthetic Peptides.- Production of Antibodies Using Proteins in Gel Bands.- Purification of Immunoglobulins Using Protein A-Sepharose.- Antibody-Enzyme Conjugate Formation.- Vacuum Blotting.- Blotting with Plate Electrodes.- Use of Dried Milk for Immunoblotting.- Immunodetection of Proteins on “Western” Blots Using 125I-Labeled Protein A.- Detection of Protein Blots Using the Avidin-Biotin System.- Detection of Protein Blots Using Enzyme-Linked Second Antibodies or Protein A.- Colloidal Gold for theDetection of Proteins on Blots and Immunoblots.- Enzyme Immobilization by Adsorption.- Enzyme Immobilization by Entrapment.- Enzyme Immobilization by Covalent Bonding.- Cell Immobilization.