Analytic selector kit

Each condition from the detergent screen “Analytic Selector Kit” (Anatrace, product number AL-SEL) (12.5 μl) was pipetted into a PCR plate and mixed with 10 μl of 2x protein buffer (without any detergent and glycerol). Protein stock (2.5 μl) was added to obtain a final protein concentration in the range of 0.1–0.5 mg/ml and thoroughly mixed by pipetting. The plate was briefly spun down in a swing-bucket centrifuge and incubated for 1 hour at room temperature prior to the thermal denaturation assay. Fluorescence and scattering of detergents without proteins were measured in 50 mM Tris, pH 7.5 and 200 mM NaCl (see Supplementary Table 2).

The differential filtration assay was performed as described by Vergis et al. [33 (link)], using the Analytic Selector kit (Anatrace). Briefy, 500 μg of full-length VanS_A was captured using a 20% slurry of His-bind resin (Millipore) in wash buffer (50 mM Tris, 500 mM NaCl, 40 mM imidazole pH 7.7) + 0.1% DDM. The slurry was dispensed into each well of a 0.2 μm filter plate, after which the beads were washed with wash buffer + 0.1% DDM, and then detergent-exchanged into the new detergents from the Analytic Selector kit. The protein was eluted from the His-bind beads and then applied directly to either a small (100 kDa) or large (300 kDa) MWCO filter plate. The eluates from these filtrations were analyzed by dot blot using an anti-6xHis antibody (Proteintech # HRP 66005); blots were scanned using a LI-COR Odyssey-FC imaging system.