Neutravidin

Flow chambers were washed with 200 µl H₂O and 200 µl phosphate-buffered saline (PBS) and subsequently incubated with 30 µl of 5 mg/ml neutravidin (Invitrogen, dissolved in PBS) for 20 min as adapted from Mieck et al. (2015) . Unbound neutravidin was removed by washing with 50 µl PBS before the glass surface was passivated for 5 min with 1% Pluronic F-127 solution (Invitrogen, dissolved in PBS) (Mieck et al., 2015 ). Subsequently, each chamber was washed with 40 µl BRB80⁺ buffer (BRB80 supplemented with 2 mM DTT, 50 mM KCl, and 20 µM taxol).

The biotin-functionalized glass slide was incubated with 20 mM PBS pH 7.4 buffer for 5 min. NeutrAvidin (Thermo Scientific, 100 nM) was incubated for another 15 min and then the slide was washed to remove unbound NeutrAvidin. The labeled protein at a concentration of 100 pM was incubated for 1 min to get isolated proteins to bind to the functionalized glass surface. The unbound proteins were then removed by washing with fresh PBS pH 7.4 buffer. This process yielded about 20 Az molecules per 20 μm × 20 μm observation area.

For the immobilization of Neutravidin (Thermo Scientific, 31000) in GeneRead flow cells, a series of cover glasses are first treated in a solution of 2.5% (3-Aminopropyl)trimethoxysilane (Sigma, 281778) in 95% aq. ethanol for 30 min at 50 °C. After cooling down, the cover glasses are removed from this solution and immediately rinsed twice with 95% aq. ethanol then once with abs. methanol. The cover glasses are then dried in an oven at 110 °C. To reduce reagent consumption, the following reaction steps are done in the flow channel of the flow cell. In order to create the flow channel of the GeneRead flow cell, flow cell base and cover glass are bound tightly together by double sided adhesive gaskets. For the generation of Neutravidin binding sites within the flow channel, each flow cell is filled with a 2 mM solution of NHS-PEG4-Biotin (Thermo Scientific, 21330) in DMSO (Merck, 1.02931) and incubated for 6 h at 50 °C. Residual amino groups are end-capped with succinic anhydride (Aldrich, 239690). After further washing steps with DMSO and PBS-T buffer (PBS supplemented by 0.05% Tween 20 and 0.515 g/l ProClin™ 300), the flow channels are incubated for 2 h with a solution of 1 mg/ml Neutravidin in PBS-T buffer. Finally, the flow cells are rinsed several times with PBS-T buffer and stored in a refrigerator at 4 °C until further use.