Single-molecule spectroscopy was performed. Leishmania cell-free lysates were prepared according to Kovtun et al., 2011; McMahon et al., 2019; Mureev et al., 2009. Where indicated, MDA-MB231 or MCF7 cells were transiently cotransfected with BRCA1-GFP and mCherry alone as the control, cavin1-Cherry, cavin2-Cherry, cavin3-Cherry, or CAV1-Cherry constructs. A PNS fraction from the MDA-MB231 and MCF7 cells was prepared in 1× PBS with protease and phosphatase inhibitors for analysis. Single-molecule coincidence measurements were performed using pairs of tagged proteins to ascertain their interaction. One protein of the pair was tagged with GFP, and the other with mCherry, and both were diluted to single-molecule concentrations (~1 nM). Two lasers, with wavelengths of 488 nm and 561 nm (to excite GFP and mCherry, respectively), were focused to a confocal volume using a 40×/1.2 NA water immersion objective. The fluorescence signal from the fluorophores was collected and separated into two channels with a 565 nm dichroic. The resulting GFP and mCherry signals were measured after passing through a 525/20 nm bandpass and 580 nm long-pass filter, respectively. The signal from both channels was recorded simultaneously with a time resolution of 1 ms, and the threshold for positive events was set at 50 photons/ms. The coincidence ratio (C) for each event was calculated as C = mCherry/(GFP + mCherry), after subtracting a 6% leakage of the GFP signal into the mCherry channel. Coincident events corresponded to ~0.25 < C < 0.75. After normalizing for the total number of events (>1000 in all cases), a histogram of the C values for the protein pair was fitted with 3 Gaussians, corresponding to signals from solely GFP (green), coincidence (yellow), and solely mCherry (red).

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