Supplementary Figure 6: Exemplary inappropriate traces for analysis.
From: Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions
(a) An exemplary trace generating false positive result. This trace shows fluctuations of small peaks occurring between 5-10 s and 30-35 s. When the threshold passes through this fluctuation, many short τoff and τon values are generated. These short τoff and τon values give a false-positive population of high kdiss and kbind value, leading to an overestimation of the kinetic values. (b) Exemplary trace with low TIR excitation intensity. Beam pattern causes inhomogeneity of the TIR excitation field, which cannot illuminate the entire field of view homogenously. Thus some molecules are excited by lower TIR excitation intensity, generating small peaks, as shown at 15 and 48 s. If the signal trace of these small peaks does not exceed the threshold, the Kinetics Analyzer program will miss these peaks and will generate longer τoff, which corresponds to small kbind. Even if the signal trace of a small peak exceeds the threshold, the trace lasts for a short time, which gives short τoff value and it result in an overestimation of kdiss. (c) Exemplary trace including a junk fluorescence molecule. Junk fluorescence molecules generally have a long lifetime, as shown between 10-30 s. This junk fluorescence molecule seems to be an autofluorescent contaminants in the PEG- cushion layer. When the trace of this junk fluorescence molecule is included in the analysis, many short τoff and τon values are generated, as shown. These values will generate a false-positive population of high kdiss and kbind values and cause the kinetic values to be overestimated.
