Phage display panning is a tool used for the discovery of novel ligands against various targets of interest. In phage display panning, you will typically start with a batch of 96 _.abi. read files, each corresponding to one well of a 96-well ELISA assay. At the end of the workflow, you will be able to view and analyze predicted 3D antibody structures and see how they dock to the corresponding antigen structure. The phage display workflow utilizes four Lasergene applications, but is fast and easy to learn and do.
Before beginning the tutorial, let’s start by reviewing the laboratory steps leading up to the .abi sequence reads. The technique (summarized in this NCBI article) begins in the laboratory with these three steps:
- Create a library of plasmids containing variable CDR loops
- Make a single-chain variable fragment (scFv) for each plasmid in the library. An scFv is a fusion protein that includes the variable regions of heavy and light chains connected by a short linker peptide about 10-25 residues in length. Leave at least a portion of vector in each sequence.
- Screen each plasmid against a specific antigen. This screening is typically done using a 96-well ELISA assay. The output is one sequence read file for each well, typically in .abi format.
Now that the laboratory portion is complete, which of the tested plasmids have an antibody sequence that is bound to an antigen? The answer can be found quickly and easily using Lasergene’s integrated phage display workflow. The tutorial is divided into four parts, with each part taking place in a different Lasergene application.
Part A. Use SeqMan Ultra to de novo assemble reads, then trim down to the target antibody translation sequence in the correct reading frame
Part B. Use SeqNinja to batch translate the sequences
Part C. Use MegAlign Pro to align sequences and create a phylogenetic tree
Part D. Use Protean 3D and the NovaApps to create a homology model of the sequence and simulate docking of the predicted antibody structures to the target epitope
Start with Part A: Assembly and trimming in SeqMan Ultra.
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