1× PBS buffer was flowed over the dechorionated embryos.
A final reaction volume of 40 µl (10 µl actin, 20 µl 2× TIRF buffer, 10 µl VASP/Lpd/etc) was flowed through a PEG/biotin-PEG TIRF flow cell, sealed with VALAP (1 1 1 mixture of Vaseline, lanoline, and paraffin wax), and imaged at 23°C.
During pulsing, depending on the load the high current is flowing through the cell buffer, resulting in thermal effects (Joule heating), which may distort or influence the experimental results.
For each measurement cycle, PBS running buffer was first flowed through the surface at a constant rate of 2 μL/s to obtain a stable baseline, and then the sample was injected at 5 μL/s for binding; after binding, the surface was washed with PBS at 2 μL/s for 300 s and regenerated with 0.5% (v/v) H3PO4 at 2 μL/s for 300 s.
Briefly, Protein G columns were equilibrated with binding buffer (Immunopure IgG, Pierce , USA after which a 1 1 mixture of sera and binding buffer was allowed to flow through under gravity, washed and eluted with elution buffer (Immunopure IgG, Pierce , USA.
m909 Fab, diluted with the running buffer, was allowed to flow through the cells.
The elution buffer was allowed to flow through the column and was collected in 1-mL fractions.
The elution buffer was then allowed to flow through the column, and every 1-mL sample was collected into a test tube containing 50 μL of neutralization buffer (fractions collected were denoted in numerical order as fractions 1, 2, 3 and so on), with the fractions being monitored with UV radiation at 280 nm.
Once the assay buffer was exited through the outlet of the flow module, the flow stopped and the changes in frequency (Δ F) and dissipation (ΔD) were recorded simultaneously.
The particles were conditioned with 0.1 M PBS buffer at pH 9.0 before 2 mL of HRP solution (prepared in the same buffer) was passed through the column at a flow rate of ∼1 mL/min.
