By Hung-Ju Chang, An-Suei Yang (auth.), Andrew E. Nixon (eds.)
Therapeutic Peptides: tools and Protocols beneficial properties organic tools for the guidance of peptide phage show libraries utilizing either filamentous and lytic phage. With contributions from popular authors within the box, the ebook additionally explores choice and screening of the ready peptide libraries for peptides with the specified functionality and the next characterization of the pointed out peptides. Written within the hugely winning Methods in Molecular Biology sequence structure, chapters comprise introductions to their respective issues, lists of the required fabrics and reagents, step by step, with ease reproducible laboratory protocols, and pointers on troubleshooting and warding off recognized pitfalls.
Practical and loyal, Therapeutic Peptides: tools and Protocols is a perfect consultant for researchers from all backgrounds looking tools for the id of healing peptide candidates.
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Additional resources for Therapeutic Peptides: Methods and Protocols
3. Elution solution: 100 mM Triethylamine (TEA; Sigma). Dissolve TEA in autoclaved distilled water immediately before use. 4. 5). 5. 5). 2 Phage Amplification and Plaque Formation Assay 1. Luria-Bertani (LB) medium: 5 g yeast extract, 10 g sodium chloride, and 10 g tryptone are dissolved in 1 l of distilled water. Autoclave and store at 4 °C. 2. NZCYM media (2×): 10 g NZ amine (casein hydrolysate), 10 g yeast extract, 10 g sodium chloride, and 4 g MgSO4·7H2O are dissolved in 1 l of distilled water.
Prepare duplicate tenfold serial dilutions of the culture in SOC medium and plate suitable dilutions onto SOC-Amp agar plates. After overnight incubation at 37 °C, count the number of colonies on the plates and calculate the number of viable cfu per vial. 4 Notes 1. Electroelution is a method that allows the recovery of highquality DNA from agarose gels. We have used the Elutrap ElectroElution System, but other electroelution systems available can also be used. 2. Due to the degenerate genetic code, certain amino acids will be present at a higher frequency than others in the displayed random peptides.
Additional beads should be prepared for negative selections. For each panning condition, 500 μl of the bead stock is subjected to the above procedure except the incubation step with biotinylated target protein. In the last washing step, the beads are divided into five 100 μl aliquots. 3 Validation of Target Protein Immobilization 1. 50 μl of beads coated with target protein are aliquoted into each well of a v-bottom 96-well plate. Typically, two wells are used per each immobilization reaction: one is used to validate the immobilization and the other is used as a negative control without antibody.