dcFCCS measurements were conducted on a home-built confocal microscope, based on a Zeiss AXIO Observer D1 fluorescence microscope equipped with solid-state 488 nm and 640 nm excitation lasers (Coherent Inc. OBIS Smart Lasers), an oil-immersion objective (Zesis, 100×, numerical aperture = 1.4) and avalanche photodiode detectors (APDs, Excelitas, SPCM-AQRH-14) as previously described.15 (link) Fluorescence passed through a pinhole (50 μm diameter), and then was split by a T635lpxr dichroic mirror (Chroma). Bandpass filters ET525/50m (Chroma) and ET700/75m (Chroma) were used to further filter fluorescence for GFP and Cy5 detection channels, respectively.
WT or mutated GFP-Rab35 protein was centrifuged at 13,000 rpm for 10 min to remove aggregates. dcFCCS experiments were carried out with 488 nm and 640 nm laser excitation at 25 °C. GFP-Rab35 and ITGα5-cyto were mixed in 20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.1% BSA and then loaded immediately onto coverslips passivated with polyethylene glycol. Raw data of photon arrival time were recorded for 5 min. Experiments were repeated three times for each experimental condition.
WT or mutated GFP-Rab35 protein was centrifuged at 13,000 rpm for 10 min to remove aggregates. dcFCCS experiments were carried out with 488 nm and 640 nm laser excitation at 25 °C. GFP-Rab35 and ITGα5-cyto were mixed in 20 mM HEPES, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 0.1% BSA and then loaded immediately onto coverslips passivated with polyethylene glycol. Raw data of photon arrival time were recorded for 5 min. Experiments were repeated three times for each experimental condition.