Figure 5. Identity of Tl intermediate.
A. Schematic of truncated peptides used as calibration standards. The number in parenthesis indicates the number of amino acids deleted from the C-terminus of the S4-5R substituted tape measure. B. Migration of standards. The constructs shown in A. were translated for 60 min (produces final full-length peptide) as described in Figure 1 and run on a NuPAGE gel (Bis-Tris 12%; lanes 1–4 loaded with 2, 2, 2, 2 µl, respectively) along with samples from a parallel translation of S4-5R (lanes 5–7 loaded with 20, 10, and 2.5 µl, respectively) using the same batch of reticulocyte lysate and identical reagents and conditions. The gel was run for a longer time to improve resolution of the bands and more accurate assessment of the mass of the paused peptide. The standards (lanes 1–4) exhibit an ascending ladder pattern with increasing mass and charge of the peptide.