Cary fluorimeter

Steady state measurements were performed with peptide-free folding reactions of K^b/β₂m. Following ultracentrifugation, different concentrations of full-length, truncated, or modified peptides were added for 5 min, and the competitive binding of 100 nM of the high affinity peptide SIINFEK_TAMRAL, labeled with carboxytetramethylrhodamine (TAMRA) on the lysine side chain, was measured by anisotropy in the Cary fluorimeter with automated polarizers.

For fluorescence-based assays, a single Msmeg colony harboring P_mft–pCherry was used to inoculate 25 ml of 7H9 media supplemented with 0.1% tyloxapol, 0.1% w/v glycerol, 50 μg/ml hygromycin, and 0.1% w/v glucose. For cultures containing OA only, 0.1% w/v OA was used. For cultures containing both OA and glucose, 0.05% w/v glucose was used, and OA was added 12 h after to the concentration of 0.05% w/v. For dose-dependent cultures containing OA (0.01%, 0.02%, 0.05%, 0.1% w/v), glucose was supplemented to the media to 0.05% w/v. The starter culture was incubated at 37 °C with shaking overnight. The overnight culture was used to inoculate 7H9 media to an absorbance of 0.05 at 600 nm (1 cm pathlength) with carbon sources supplemented as described previously. The cultures (30 ml) were incubated at 37 °C with shaking for 40 h. Aliquots were taken every 2 h, and absorbance at 600 nm values were measured. To track the expression of the P_mft–pCherry fusion, fluorescence measurements were taken on a Cary Fluorimeter every 2 h using an excitation of 585 nm and an emission of 612 nm.