t50: time after which 50% of the protein has become insoluble

Stability/Folding

time after which 50% of the protein has become insoluble

min

SDS-PAGE , Thermal Denaturation , Phosphoimage

None

38°C

None

7.5 μM

None

None

Isolated VH domains were brought to a concentration of 7.5 μM in PBS, 100 mM beta-mercaptoethanol. To determine the time dependence of thermal denaturation, 50 mL aliquots were incubated at 38°C for variable times, cooled on ice for 5 s, and centrifuged at 18.000 rcf, 4°C, for 5 min. To 30 μL of the soluble supernatant, 10 μL of a BSA solution of 0.5 mg/mL was added as an internal standard. Samples were run on a 12.5% SDS-PAGE gel, stained with Sypro Red (FMC Bioproducts, Rockland, Maine), and the bands were quantified on a phosphorimager (Molecular Dynamics, Sunnyvale, California). To determine the temperature dependence of thermal denaturation, samples were incubated for 1 h at temperatures from 35 to 44°C in steps of ~1°C, and the remaining soluble material was quantified as above.