Bsa biotin

Slides were incubated with wash buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mM EGTA, 1 mM DTT, 1% pluronic) for 5 min. Samples were settled onto the coverslip for 25 min before imaging. Imaging was performed using a Nikon Ti-E Eclipse microscope equipped with a 100X 1.49 N.A. Plan Apo oil immersion objective (Nikon). The samples were excited in near-TIRF using 488, 561, and 633 nm laser beams (Coherent). The emission signal was passed through a filter wheel and detected by Andor Ixon EMCCD Camera (512x512 pixels). The effective pixel size was 106 nm after 1X magnification and 160 nm after 1.5X magnification. Image processing is described in the “Quantification and Statistical Analysis” section.
For experiments involving DNA bound to the surface of the slide, chambers were incubated with 1 mg/mL Biotin-BSA (Sigma-Aldrich, 9048-46-8) for 2 min, incubated with 1 mg/mL streptavidin (Thermo Fisher Scientific, 434301) for 2 min, washed twice with 20 μL wash buffer, incubated with 1 μM biotinylated 8ds3ss DNA (IDT) for 2 min and washed twice with 20 μL wash buffer. Shelterin droplets were formed in a test tube, flowed into the chamber, and settled on the coverslip for 10 min. 5 μL solution containing the protein or DNA being tested was introduced to the chamber, and the sample was imaged using time-lapsing for 1 s every 10 s for 1 hr.

GUVs with a plasma membrane-like lipid composition consisting of 30 mol% Chol, 15 mol% SM, 34 mol% PC, 10 mol% PE, 5 mol% PS, 5 mol% PI, and 1 mol% Biotinyl-PE (Avanti Polar Lipids) were generated based on electro-swelling using platinum electrodes (García-Sáez et al., 2009 (link)). GUVs were supplemented with either PI(4,5)P₂ or a Ni-NTA lipid at 2 mol% at the expense of PC as indicated. For visualization either 0.05 mol% rhodamine B-labeled PE for FGF2 translocation assays or 0.002 mol% Atto-633-labeled dioleolyl-PE (Atto-633-DOPE, ATTO-TEC) for Dual-color FCS measurements was added. The dried lipid film was hydrated with a 300 mM sucrose solution (300 mOsm/kg, Wescor Vapro). Where indicated, long-chain heparins (50 mM; based on disaccharide units) were included in the lumen of GUVs in order to mimic heparan sulfates. Swelling was conducted at 45°C (10 Hz, 1.5 V for 50 min [without heparin] or 70 min [with heparin], 2 Hz, 1.5 V for 25 min). In order to remove excess amounts of heparin and sucrose, GUVs were gently washed twice with buffer B (25 mM HEPES pH 7.4, 150 mM NaCl, 310 mOsmol/kg) and collected via centrifugation (1200 × g; 25°C; 5 min). Imaging chambers (LabTek for FGF2 translocation assays, ibidi for Dual-color FCS) were incubated sequentially with 0.1 mg/mL Biotin-BSA (Sigma A8549) and 0.1 mg/mL Neutravidin (Thermo Fisher Scientific A2666) in buffer B.