Thermostability shift assay and library screening

1.     A 5 mg ml⁻¹ stock of CPM dissolved in DMSO was diluted 50-fold into assay buffer, which can be the detergent/lipid containing buffer of the purified membrane proteni.  For example, 20 mM HEPES-NaOH pH 8.0, 150 mM NaCl, 0.1% dodecyl maltoside or lauryl maltose neopentyl glycol supplemented with 0.1 mg ml^-1 lipid in the case of mitochondrial carriers.

2.     This working stock was vortexed and allowed to equilibrate in the dark at room temperature for 10 min. Pre-incubation of CPM with detergent and protein:detergent:lipid micelles is an essential step to avoid significant drift in baseline fluorescent signal during melting. CPM differentially partitions into the micelles, which needs to proceed to equilibrium, otherwise changes in fluorescence during the run are observed due to equilibration of the dye rather than thermal denaturation of the protein.

3.     One to four micrograms of protein were added into a final volume of 45 μl of assay buffer in 200 μl thin-walled PCR tubes, and 5 μl CPM working solution was added.

4.     The solution was vortexed and allowed to equilibrate in the dark for a further 10 min.

5.     PCR tubes containing the reaction mixture were transferred to the 36-position rotor and loaded into the Qiagen RotorGene Q. Other qPCR machine with appropriate filters can be used, but Rotorgene Q can do 36 or 72 different samples and rotates, which prevent the interference of bubbles.

6.     An initial pre-incubation step of 90 s was set to allow the temperature to equilibrate to 25 °C.

7.   Measurements were made in 1 °C intervals from 25 to 90 °C with a ‘wait between reading’ set to 4 s, which equated to a ramp rate of 5.6 °C min⁻¹.

8.     Data were analyzed and melting temperatures (the inflection point of the melting curve) were determined with the software supplied with the instrument.

9.     Raw and analyzed data were exported from the software and Prism (Graphpad; www.graphpad.com) was used to interpret the results graphically.

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