Heterogeneity of the respiratory syncytial virus 22K protein revealed by Western blotting with monoclonal antibodies.
Respiratory syncytial (RS) virus-infected HeLa, HEp-2, Vero and BS-C-1 cell lysates were electrophoresed on SDS-polyacrylamide gels under reducing conditions and analysed by Western blotting and immunoperoxidase using monoclonal antibodies specific for the 22K protein (relative mol. wt. of 23,000 in our gel system). Three novel polypeptides with mol. wt. of 24,000, 21,000 and 17,000 were stained in addition to the 23,000 polypeptide which was present in the greatest amount in all three virus strains tested regardless of host cell line. When samples were electrophoresed under non-reducing conditions each of the three higher mol. wt. polypeptides seen in reducing gels migrated as two bands (total of six bands) with altered electrophoretic mobilities. In experiments using the alkylating agent iodoacetamide under conditions where the novel 24,000, 21,000 and 17,000 polypeptides were not visible, the number of mobility variants of the 23,000 polypeptide which could be detected in non-reducing conditions was increased from two to four. At least one, and possibly three, of these variants was the result of conformational variation in the 23,000 polypeptide caused by the generation or rearrangement of intrachain disulphide bonds after the infected cells were lysed in SDS-PAGE sample buffer. Post-lysis conformational changes were minimized by treatment of the infected cells with iodoacetamide before solubilization or by decreasing the SDS concentration or using milder detergents in the lysis buffer.
D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007460:Iodoacetamide / Q000494:pharmacology; D008970:Molecular Weight; D011487:Protein Conformation; D012136:Respiratory Syncytial Viruses / Q000032:analysis*; D014764:Viral Proteins / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D004220:Disulfides; D014764:Viral Proteins; D007460:Iodoacetamide