FIGURE 4.

Effects of protein concentration on VHH denaturation and analysis of the molecular weight of heat-inactivated anti-hCG VHH by gel permeation chromatography and SDS-PAGE. A, VHH with different concentrations in HBS-EP buffer were subjected to 40 cycles of heating-cooling cycles (unfolding at 90 °C for 5 min and refolding at 20 °C for 5 min). Error bars represent S.D. B, room temperature gel permeation chromatography profiles of VHH without heat treatment, with 40 cycles of heating-cooling cycles, and with continuous incubation at 90 °C for 1,600 min (m) at a protein concentration of 10 μm. Arrowheads indicate the elution volumes of serum albumin (67 kDa), ribonuclease A (13.7 kDa), and trypsin inhibitor (6.5 kDa). C, VHH (10 μm) was treated with continuous incubation at 90 °C for 1,600 min and analyzed using a polyacrylamide gradient gel (5–20%) with or without reduction with 10 mm dithiothreitol. The numbers on the left of the panel indicate the molecular weights of marker proteins. mAU, milliabsorbance units.