Journal: PLoS ONE
Article Title: Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes
Figure Lengend Snippet: Characterization of anti-N scFv-phages. (A) Western protein analysis of scFv-phage binding to recombinant SCoV-N protein. Purified His 6 -SCoV-N recombinant protein was separated on gradient sodium dodecyl sulfate (5% - 20%) polyacrylamide gel and transferred onto a 0.2 µm nitrocellulose membrane. The stripes were probed with different anti-N scFv-phages, including L9A6B, L9A11B, L4A3A, L4A3B, L4A8B, and M13KO7 helper phage (negative control) as indicated. Purified SCoV-N protein was stained with Coomassie Blue for reference. (B) Cross-reactivity of selected anti-N scFv clones was evaluated using phageELISA against different antigens, including BSA, SCoV-N, insulin, thyroglobulin, Ang II-BSA conjugate, LPS, ssDNA as indicated. Error bars represent SEM of three separate experiments each performed in duplicate. Difference between means of binding to SCoV-N protein versus to other unrelated antigens is evaluated by One Way ANOVA using GraphPad Prism 4. Statistical significance is indicated as *** ( P
Article Snippet: Subsequently, log-phase TG1 transformants were super-infected with M13KO7 helper phage (GE Healthcare) in a multiplicity of infection (moi) ratio of 3∶1, and phages were rescued at 30°C overnight with gentle shaking.
Techniques: Western Blot, Binding Assay, Recombinant, Purification, Negative Control, Staining, Clone Assay