Monitoring SCCH Domain Interactions

Microscale thermophoresis (MST) analyses of the SCCH domain:polyalanine peptide interaction were carried out using our Monolith NT.115 (Nano Temper Technologies). A peptide containing a tandem of 7 alanine residues was labeled with Cy5 (GL Biochem Shanghai Ltd.), and was used at constant final concentration of 100 nM. The labeled peptide was mixed with the ligand, a purified recombinant unlabeled UBA6 (623-889) or UBA1 (624-891) SCCH domains. 1:2 serial dilutions of the ligands from 200 µM to 1.56 µM final concentration were assayed. After a short incubation, the samples were loaded into premium glass capillaries before data collection. All MSTs were performed twice at Excitation Power 30% of the LED and MST power of 20% and 40%.