Immunology
A review of the earlier chapters in this book documents the importance of immunological methods to the purification of proteins as well as to the identification of specific cDNA clones.
Abstract
C ertain technological advances in the field of molecular biology were made possible in part by earlier progress in the field of immunology. A review of the earlier chapters in this book documents the importance of immunological methods to the purification of proteins as well as to the identification of specific cDNA clones. Specific antibodies have greatly facilitated the purification of proteins by immunoaffinity chromatography (UNIT 10.11) and immunoprecipitation (UNIT 10.16). One limitation of immunoaffinity chromatography has been that the harsh dissociation conditions required to elute bound antigens from high-affinity antibodies sometimes denature the eluted antigens. UNIT 11.18 presents a method to circumvent this problem by utilizing polyol-responsive antibodies that release their bound antigens under gentle dissociation conditions, employing a combination of various low molecular weight polyhydroxylated compounds (e.g., ethylene glycol) and nonchaotropic salts (e.g., ammonium sulfate). These polyol-responsive antibodies can be readily identified and isolated from typical fusions, prepared by standard hybridoma procedures. When pure protein has been unavailable for deducing the complementary oligonucleotide sequence, specific antibodies have been utilized to screen recombinant DNA libraries for the desired cDNA clones (UNIT 6.7) and selected mRNA for the translation of desired protein (UNIT 6.8). Specific antibodies have also been utilized to identify antigen by western blotting (UNIT 10.8).