Figure 2. Optimization of translation efficiency and
confirmation of mRNA/cDNA-peptide conjugates. Gel images of the
mRNA-peptide conjugate formation under various translation conditions.
(A) Difference in length of translation before salt addition. Number
indicates different incubation time (in minutes) carried out at 37
degrees C. (B) Difference in salt concentrations with original
concentration (1x) as 750 mM KCl, 65 mM MgCl2 used in
the previous studies (Naimuddin et al., 2011; Naimuddin & Kubo, 2016)
(C) Difference in lengths of incubation time (in minutes) after salt
addition. (D) Trypsin digestion of mRNA-tag and mRNA-peptide conjugate
products. (E) Detection of FITC fluorescence signal from the mRNA-tag,
mRNA-peptide conjugate, and mRNA/cDNA-peptide conjugate products (left)
and detection of FLAG epitope sequence using chemiluminescent Western
blotting (right) from the same membrane.