In vitro translation using cell-free system for mRNA display
Two μg of the mRNA-tag sample was used for in vitro translation using PUREfrex 1.0 (Gene Frontier) in the 100 µl reaction mix. The optimized condition for efficient mRNA-peptide conjugate formation are as follows: initial translation at 37 ℃ for 30 min, followed by addition of a salt mix to a final concentration of 32.5 mM MgCl2and 375 mM KCl, and then a second incubation at 37 ℃ for 60 min. After this step, an equal volume of 2x Laemmli sample buffer (Bio-Rad) was added, mixed and centrifuged at 10,000 x g, 1 min at room temperature to remove the salt precipitant. The supernatant containing the translated product was recovered and further resolved on a 3.5% stacking - 10% resolving polyacrylamide SDS-PAGE gel at constant 50 mA. 8 M urea was added to the resolving gel only. Afterwards, the gel band corresponding to mRNA-peptide conjugate was excised, purified using the Electro-Eluter. and precipitated with ethanol, as described in the previous section.