Ezrin T567D was recombinantly
expressed in E. coli (BL21(DE3)pLysS,
Novagen, Madison, WI, USA) and purified as described previously.33 (link) RIfS was used to measure the formation of SLBs
on the silicon wafers and binding of the protein onto the membranes.
RIfS is a noninvasive label-free technique to determine optical layer
thicknesses (OT = nd). OT values were monitored using
a flame-S-UV/vis spectrometer (Ocean Optics, Dunedin, FL, USA), recording
a spectrum every 2 s and analyzed utilizing a custom MATLAB script
(R2021a, Mathworks). The experimental setup was described previously.37 (link) After SLB formation, the membrane surface was
rinsed with ezrin buffer and a BSA solution (1 mg/mL in ezrin buffer)
for 5 min. After rinsing again with ezrin buffer for 5 min, ezrin
T567D was added (0.8 μM) for 10 min. Unbound protein was removed
by rinsing with ezrin buffer.
expressed in E. coli (BL21(DE3)pLysS,
Novagen, Madison, WI, USA) and purified as described previously.33 (link) RIfS was used to measure the formation of SLBs
on the silicon wafers and binding of the protein onto the membranes.
RIfS is a noninvasive label-free technique to determine optical layer
thicknesses (OT = nd). OT values were monitored using
a flame-S-UV/vis spectrometer (Ocean Optics, Dunedin, FL, USA), recording
a spectrum every 2 s and analyzed utilizing a custom MATLAB script
(R2021a, Mathworks). The experimental setup was described previously.37 (link) After SLB formation, the membrane surface was
rinsed with ezrin buffer and a BSA solution (1 mg/mL in ezrin buffer)
for 5 min. After rinsing again with ezrin buffer for 5 min, ezrin
T567D was added (0.8 μM) for 10 min. Unbound protein was removed
by rinsing with ezrin buffer.
Free full text: Click here
