User:1974manish/NAPPA

Nucleic acid programmable protein array:

This system was first developed by LaBaer and colleagues in 2004 by using in vitro transcription and translation system. They use DNA template encoding the gene of interest fused with GST protein, and it is immobilized in the solid surface. Anti-GST antibody and biotinylated plasmid DNA are bounded in aminopropyltriethoxysilane (APTES)-coated slide. BSA can improve the binding efficiency of DNA. Biotinylated plasmid DNA is bound by avidin. New protein is synthesized by using cell-free expression system i.e. rabbit reticulocyte lysate (RRL), where the protein is captured through GST tag by bounded antibody on slide. To test protein-protein interaction, the targeted protein cDNA and query protein cDNA are immobilized in coated slide. By using the in vitro transcription and translation system, targeted and query protein is synthesized in a same extract. The targeted protein is bound to array and query protein is used to probe the array. The query protein is tagged with hemagglutinin (HA) epitope. Thus, the interaction of proteins can be visualized with the antibody against HA