Western blot analysis
Western blot analysis was performed to confirm the physical linkage of
peptides and their cognate mRNA sequences in the form of mRNA-peptide or
mRNA/cDNA-peptide conjugates. A control mRNA sequence (Supplementary
Table 1) encoding a FLAG epitope (DYKDDDDK) was ligated with
puromycin-FITC DNA tag and translated under optimized condition (see
previous section). Samples were run on a 10% polyacrylamide SDS-PAGE
gel at constant 30 mA until the samples stack and reach the separating
gel, and at constant 50 mA for additional 40 minutes. The samples in the
gel were semi-dry transferred to a PDVF membrane (Bio-Rad) at constant
110 mA for 60 minutes. Blocking was carried out using the PVDF Blocking
Reagent for Can Get Signal (Toyobo) and, the membrane was incubated
using Anti DYKDDDDK tag monoclonal antibody peroxidase-conjugated
(FUJIFILM Wako Pure Chemical) and Can Get Signal, solution 2 (Toyobo)
with a 1: 12,000 ratio, respectively. The membrane was then revealed
with chemiluminescence using ECL Select Western Blotting Detection
Reagent (GE Healthcare).