Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots.
An affinity-purification method has been developed for the rapid, efficient, and precise elution of antibodies specifically bound to antigens immobilized on nitrocellulose after blot transfer from SDS polyacrylamide gels. The applicability of this technology has been demonstrated using antisera raised against the nuclear matrix-pore complex-lamina fraction prepared from Drosophila melanogaster embryos. In so doing, we have established the existence in whole embryo lysates, of two nearly identical forms of the predominant 74-kilodalton polypeptide previously identified in lower resolution studies of the nuclear matrix-pore complex-lamina fraction. These species, distinguishable on the basis of a slight difference in SDS PAGE mobilities on low concentration polyacrylamide gels, are immunochemically cross-reactive and have been localized exclusively to the nuclear periphery (nuclear envelope) by indirect immunofluorescence analyses of cryosections. The steady-state levels of these two polypeptides have been examined in total embryo lysates both as a function of embryogenesis and in response to heat shock. The larger species is not detectable in early embryos but approaches levels approximately equal to that of the smaller form by about the temporal midpoint of embryonic development. In response to heat shock, this larger form appears to be converted nearly quantitatively into the lower molecular weight polypeptide. These results, as well as the general reliability of the nitrocellulose blot immunoaffinity-purification methodology, have been substantiated through the use of monoclonal antibodies.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D003101:Collodion; D004331:Drosophila melanogaster / Q000196:embryology*; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006358:Hot Temperature; D007074:Immunoglobulin G / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000032:analysis*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D008565:Membrane Proteins; D003101:Collodion