Author: Wilton T. Snead; Wade F. Zeno; Grace Kago; Ryan W. Perkins; J Blair Richter; Chi Zhao; Eileen M. Lafer; Jeanne C. Stachowiak
Title: BAR scaffolds drive membrane fission by crowding disordered domains Document date: 2018_3_4
ID: drqseaaa_68
Snippet: The biotinylated PLL-PEG was made according to a previous protocol (Ruiz -Taylor et al., 2001) . Briefly, amine-reactive PEG-SVA (succinimidyl valerate) and biotin-PEG-SVA was added to a 40 mg mL -1 mixture of PLL in 50 mM sodium tetraborate pH 8.5 at a molar ratio of one PEG per five lysine subunits. PEG-biotin comprised 2% of the total PEG amount. The mixture was stirred continuously for 6 h at room temperature and buffer exchanged into PBS usi.....
Document: The biotinylated PLL-PEG was made according to a previous protocol (Ruiz -Taylor et al., 2001) . Briefly, amine-reactive PEG-SVA (succinimidyl valerate) and biotin-PEG-SVA was added to a 40 mg mL -1 mixture of PLL in 50 mM sodium tetraborate pH 8.5 at a molar ratio of one PEG per five lysine subunits. PEG-biotin comprised 2% of the total PEG amount. The mixture was stirred continuously for 6 h at room temperature and buffer exchanged into PBS using Centri-Spin size exclusion columns (Princeton Separations). Imaging wells were made by placing silicone gaskets onto ultraclean coverslips. Wells were coated for 20-30 min with biotinylated PLL-PEG diluted tenfold in 20 mM MOPS pH 7.35, 150 mM NaCl buffer. After coating, the well was washed repeatedly with MOPS buffer to wash out excess PLL-PEG. Neutravidin was added to the well following the same process as for PEG-silane slides.
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