Alkaline phosphatase conjugated protein A as a sensitive reagent to immunoscreen an expression cDNA plasmid library: isolation of cDNA to the calcium-binding protein of the chick embryonic chorioallantoic membrane.
A highly efficient immunoscreening procedure has been developed to isolate cDNA clones to the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM). A library of total CAM cDNA was constructed using the expression plasmid vector, pUC 19. Bacterial clones containing plasmids with CaBP cDNA inserts were detected immunohistochemically based on their expression of hybrid CaBP protein sequences. For immunodetection, nitrocellulose bacterial colony replicas were treated with specific antibodies to the CaBP followed by incubation with Staphylococcus aureus Protein A conjugated with alkaline phosphatase (AP) which served as a secondary immunoreagent. Positive clones were then histochemically identified based on AP enzyme activity. The identity of the immunopositive clones was further verified by in vitro translation of mRNA selected by hybridization to the cloned cDNA. The AP-based immunoscreening procedure yields stable reaction products with relatively low background, and should find general application for isolating specific cDNA clones from expression cDNA libraries.
D000469:Alkaline Phosphatase; D000482:Allantois; D000818:Animals; D017317:Annexin A6; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002642:Chick Embryo; D002823:Chorion; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D007120:Immunochemistry; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012333:RNA, Messenger; D013205:Staphylococcal Protein A
D017317:Annexin A6; D002135:Calcium-Binding Proteins; D007202:Indicators and Reagents; D012333:RNA, Messenger; D013205:Staphylococcal Protein A; D004247:DNA; D000469:Alkaline Phosphatase