Protein Thermal Shift (PTS) assay
Protein Thermal Shift assay was used to assess the thermal stability
change of purified VCP and VCP fragment protein in the presence of
gossypol acetate. 3μg 6XHis-VCP Full length protein or 6XHis-VCP
fragment protein were incubated with 5μM Ace.gos in ice for 30min (100mM
Tris-HCl pH 7.5, 150mM NaCl), and then heated under a set of temperature
gradient (50/56.8/64.3/72.0/64.3/72.0 ℃) using the T100 Thermal Cycler
PCR instrument. After heating, samples were centrifuged at12000 rpm for
2min at 4℃. The supernatant were transferred and kept for detection of
the retained soluble protein by Coomassie blue staining. The strength of
the bands was obtained by gray scale scanning by Image J, and the Tm
value was calculated by Boltzmann fitting using GraphPad Prism 6.