Immunological screening of standard cDNA libraries in pBR322 vectors: detection of human fibrinogen and prothrombin cDNA clones.
The in situ immunological detection of antigens encoded by cDNA inserted into the PstI site of pBR322 plasmids was optimized. It was found that sensitivity of the detection was dramatically increased by in situ amplification of the recombinant plasmids on chloramphenicol-containing medium followed by a brief incubation without chloramphenicol during which protein synthesis resumes. In addition, several modifications of the previously described methods which permit total suppression of background and false positives are described. These techniques allowed easy detection of cDNA clones for human B beta- and gamma-fibrinogen and -prothrombin using a human liver double-stranded cDNA recombinant plasmid library in pBR322 vectors.
D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA; D004274:DNA, Recombinant; D004926:Escherichia coli; D005340:Fibrinogen / Q000235:genetics*; D006801:Humans; D007120:Immunochemistry; D008099:Liver / Q000378:metabolism; D010957:Plasmids; D011516:Prothrombin / Q000235:genetics*
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011516:Prothrombin; D005340:Fibrinogen; D004247:DNA