Editing S30 E. coli extract (section)

=== In Vitro Coupled Transcription–Translation and RIA. ===

After the fifth round of ribosome display, PCR products were cloned into the vector pTFT74 (20). In vitro coupled transcription–translation in the S-30 E. coli system was performed using 50 μg/ml plasmid DNA and conditions similar to those described above with the following modifications. A coupled transcription–translation was carried out for 30 min at 37°C, and the reaction mixture was supplemented with T7 RNA polymerase at 2000 units/ml and 0.5 mM UTP and TTP. The mixture contained [35S]methionine at 50 μCi/ml (1 μCi = 37 kBq) and each amino acid except methionine at 0.35 mM. After translation, the reaction mixture was diluted 4-fold with PBS and bound to immobilized hemagglutinin peptide in a microtiter well. After 60 min of incubation with gentle shaking, microtiter wells were washed five times with PBS containing 0.1% Tween 20, and bound radioactive protein was eluted with 0.1 M triethylamine. Eluted protein was quantified in a scintillation counter.
[[Category:Antibodies]]