TIRFM experiments were conducted
at 22 ± 0.5 °C on a Nikon A1R TIRF instrument and a home-built,
objective-based TIRFM instrument as described previously.57 (link)−59 (link) Supported lipid bilayers in physiological buffer [140 mM KCl, 15
mM NaCl, 0.5 mM MgCl, 26 μM CaCl2, 20 μM EGTA,
5 mM reducedl -glutathione, and 25 mM HEPES (pH 7.4)] were
imaged before and after sequential additions of buffer, BSA, and fluorescent
protein. Typically, only a few dim, rapidly dissociating fluorescent
contaminants were observed on the bilayer prior to the addition of
protein. After protein addition, samples were allowed to equilibrate
for 5 min. Then, to minimize contributions from small numbers of immobile
fluorescent particles (presumably inactive protein aggregates), a
bleach pulse power ∼30-fold higher than that used for imaging
was applied for ∼5 s, and the fluorescence was allowed to recover
for 60 s before data were acquired. For each sample, a set of three
or four movie streams were acquired at a frame rate of 20 frames/s,
and a spatial resolution of 6.3 pixels/μm on the Nikon A1R instrument
or 4.2 pixels/μm on the home-built instrument, using NIS Elements
Basic Research (Nikon). Subsequent particle tracking analysis was
conducted using ImageJ,64 (link) and data processing
and fitting were conducted using Mathematica (Wolfram Research) and
GraphPad Prism 5 (GraphPad Software, Inc.).
at 22 ± 0.5 °C on a Nikon A1R TIRF instrument and a home-built,
objective-based TIRFM instrument as described previously.57 (link)−59 (link) Supported lipid bilayers in physiological buffer [140 mM KCl, 15
mM NaCl, 0.5 mM MgCl, 26 μM CaCl2, 20 μM EGTA,
5 mM reduced
imaged before and after sequential additions of buffer, BSA, and fluorescent
protein. Typically, only a few dim, rapidly dissociating fluorescent
contaminants were observed on the bilayer prior to the addition of
protein. After protein addition, samples were allowed to equilibrate
for 5 min. Then, to minimize contributions from small numbers of immobile
fluorescent particles (presumably inactive protein aggregates), a
bleach pulse power ∼30-fold higher than that used for imaging
was applied for ∼5 s, and the fluorescence was allowed to recover
for 60 s before data were acquired. For each sample, a set of three
or four movie streams were acquired at a frame rate of 20 frames/s,
and a spatial resolution of 6.3 pixels/μm on the Nikon A1R instrument
or 4.2 pixels/μm on the home-built instrument, using NIS Elements
Basic Research (Nikon). Subsequent particle tracking analysis was
conducted using ImageJ,64 (link) and data processing
and fitting were conducted using Mathematica (Wolfram Research) and
GraphPad Prism 5 (GraphPad Software, Inc.).
