Again, 500 µL of RPE buffer was pipetted into the tubes, and the columns were centrifuged for 15 seconds at 10,000×g and placed in new 2 ml catch tubes and spun at full speed for one minute to ensure that the membranes were dry.
A volume of 100 μl phosphate buffer was pipetted into the ring on the upper side (Fig. 1).
To ensure that no beads have been residing in the deep well plate, 400 μl wash buffer is pipetted into the plate and a transfer to the filter plate is carried out as described above.
One 250 μg aliquot of actin was diluted to 1 mg/ml with 225 μl of ATP-supplemented general actin buffer, mixed to ensure complete re-suspension and left on ice for 30 min. 25 μl of actin polymerization buffer was pipetted into the actin protein and mixed well.
Aliquots of 50 μL of the 1-octanol phase and 500 μL of the phosphate buffer phase were pipetted into separate tared Eppendorf tubes with adequate care to avoid cross contamination between the two phases.
For each sample, lysates, neat plus four serial dilutions (dilution factor 0.8) and one buffer control, were pipetted into V-shaped ABgene 384-well plates (Thermo Fisher Scientific, Rockford, IL, USA).
For DLS, reactions of SbpA in self-assembly buffer (100 μL) were pipetted into a disposable solvent resistant microcuvette (Malvern Instruments, Worcestershire, United Kingdom), sealed with a plastic lid and PARAFILM, and then placed into the instrument.
In brief, 100 μl of diluted standards, quality controls, dilution buffer and diluted samples, were pipetted into a 96-microtitre plate in duplicate.
All buffers, peptides, and RNA were pipetted into wells via a liquid handling robot (Janus, PerkinElmer).
Forty-µl samples containing 1 mg/ml SOD1 (65 µM polypeptide chain or 33 µM dimer) in buffers containing 10 µM ThT were pipetted into wells of a Corning Costar 384-well microplate with transparent bottoms, white walls, and non-binding surface (Corning 3653).
