Gibco 0.25 w v trypsin phenol red

Before evaluating the neutralization potential of design variants, proteins were tested for its susceptibility to proteases as the cell-based neutralization assay contains high amounts of trypsin. Cleavage can occur either if a site is accessible in a loop area of if the protein is partially unfolded and exposes a site. Proteins to be tested were diluted to 1mg/mL in PBS to which trypsin was added with a final concentration of 0.005% (w/v) (Gibco 0.25% (w/v) trypsin/phenol red, Life Technologies, Carlsbad, CA) and incubated at 37°C. Time points were taken by removing 6 μL aliquots of solution. Digestion was instantly quenched by mixing with 6 μL of 2× Laemmli Sample Buffer (Bio-Rad) and incubating it for 2 min at 95°C. Samples were loaded onto a 12% NuPage Bis-Tris gel (Life Technologies, Carlsbad, CA) and run 1× MES buffer (50 mM MES, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.3). Trypsin did not degrade monomeric or trimeric constructs over the monitored period of 24 h at 37°C.