Low binding tubes

All protease digestions were performed in 96FASP plates with MWCO 10 kDa (Acroprep Advance^TM) by adapting a 96FASP sample preparation protocol for protease digestion under native conditions^{28 (link),29 (link)}. The first step was to wash the filter units to remove any residuals. For this 100 µl of 20 mM Ammonium bicarbonate pH 7.8 were added to the wells and the plate was centrifuged at 1,300 g for 10 min before repeating this step once more. Afterwards, native cell lysate standardized in 20 mM Ammonium bicarbonate pH 7.8 was added at a final 50 µg of total protein per well and mixed with the investigated proteases at 1/50 [E]/[S] ratio. The samples were incubated at 37 °C for 4 h and collected by a 15 min centrifugation at 1,300 g in a low binding 96-well conical plate. The collection step was repeated by adding 100 µl of MS-grade water. The fractions were transferred to low-binding tubes (Eppendorf) and concentrated on the SpeedVac to complete dryness. The samples were stored at −80 °C until analysis. Before analysis, the samples were re-suspended in 20 µl of MS-grade water with 0.1% formic acid and the peptide concentration was determined with Nanodrop UV spectrometer. The sample concentration was adjusted to 1 µg/µl with water containing 0.1% formic acid.

Polystyrene microspheres coated with Streptavidin (mean diameter 4.95 μm) were purchased from Bangs Laboratories (US). Washes and incubations were preferably performed in low-binding tubes (Eppendorf, Germany). Beads were washed and centrifuged three times for 3 min at 21,000 g in 100 μl of binding buffer (10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, 0.0005% TritonX-100). Beads were then resuspended in 10–50 μl of binding buffer containing 5 to 300 pmol of template DNA. Reactions were incubated at room temperature for 3 h or overnight under continuous mixing using a MonoShake Microplate Mixer (Variomag) at maximum speed (2000 rpm). Following binding, beads were centrifuged for 3 min at 21,000 g and the supernatant with the unbound DNA was removed. The amount of DNA bound on the beads was estimated by measuring the DNA concentration in the supernatant using a Nanodrop photospectrometer (ThermoFisher, US). Beads were repeatedly washed with binding buffer and nuclease-free water until the DNA concentration in the supernatant was close to zero. DNA-immobilization on magnetic beads (Dynabeads M-280, mean diameter 2.8 μm, Thermofisher, US) was performed following the same protocol.

In order to determine the surrogate neutralization ability of NoV GII.4-specific antibodies, a blocking assay was conducted according to a published protocol [24 (link)] using human saliva type A as the source of HBGAs [42 (link)]. Individual mouse sera (diluted 1 : 50) or groupwise pooled sera (titrated from 1 : 50) were preincubated with 0.1 μg/ml GII.4 VLPs for 1 h at +37°C in low-binding tubes (Eppendorf, Hamburg, Germany) prior to adding the preparations on saliva-coated plates. The bound VLPs were detected using human NoV-positive serum (1 : 4000) and horseradish peroxidase- (HPR-) conjugated anti-human IgG antibody (1 : 6000, Novex, Invitrogen) reacting with OPD substrate (Sigma-Aldrich). The OD readings were measured as described above. Wells incubated with VLPs lacking mouse sera were added to each assay to determine the maximum binding OD. The blocking index (%) was calculated as 100% − [(OD wells with VLP‐serum mix/maximum binding OD) × 100%]. The results are expressed as the mean blocking indexes of individual mice for each immunization group or as mean blocking indexes of replicate wells if groupwise pooled sera were used.

In order to visualize TDP‐43 (wt, 5D, 12D and 12A) aggregates formed under the above described assay conditions, 10 µM Al.488‐labeled TDP‐43‐MBP‐His₆ was set up in low binding tubes (Eppendorf) in aggregation buffer and incubated with or without 100 µg/ml His₆‐TEV protease. Samples were shaken at 1,000 rpm at RT for 30 min and then transferred into a 384‐well black plate (Greiner Bio‐One), incubated at RT and imaged by confocal microscopy after 2, 8 and 24 h.

Stock solutions for the individual peptides, Aβ1-40 and Aβ1-42, were purchased as lyophilized white powder with a peptide purity of over 95 %. The stock solutions were reconstituted with DMSO to achieve a final concentration of 1.00 mg/ml. Aliquots of 20 µL were prepared and stored at − 20 °C in polypropylene vials (Low Binding tubes, Eppendorf, Germany). On the day of usage, calibration and Quality Control (QC) samples were prepared gravimetrically. Calibration solutions were made by diluting stock solutions with water/acetonitrile/NH₄OH (49:50:1) to obtain different concentrations of Aβ1-40 and Aβ1-42 standards, which included 7.5, 15, 31.125, 62.5, 125, 250 ng/ml. QC samples were prepared in a similar manner at concentrations of 25, 75, and 150 ng/ml. For the creation of working check solutions, the Aβ1-40 and Aβ1-42 standards were spiked with an equal volume of labeled internal standards ([15N] Aβ1-40 and [15N] Aβ1-42) at a concentration of 75 ng/mL each. Matrix solutions were prepared by adding 20 μL of standard and 20 μL of labeled standard to 180 μL of surrogate CSF containing 4 mg/ml bovine serum albumin (BSA).
The final concentrations of the working standard solutions were 3.5, 7, 15, 30, 60, and 115 ng/mL. The working QC solutions had concentrations of 15, 30, and 75 ng/ml. Prior to LC/MSMS analysis, the spiked solutions were purified via the SPE method.

Double emulsions were resuspended with a 200 μL
pipet prior
to measuring. Flow cytometric analysis was carried out on a CytoFLEX
S machine for double emulsions stored in TAB (1.5% tween). Aptamer-Cy5
fluorescence was quantified using 640 nm excitation, with a 660/10
nm bandpass filter. Flow cytometric sorting of double emulsions was
performed on FACSAria III or FACSAria II instruments (BD) , with sorting
into different low-binding tubes (Eppendorf) containing 100 μL
of nuclease-free water according to aptamer fluorescent intensity.
Prior to sorting, the double emulsions were often diluted ∼5
times in TAB (1.5% tween). Cy5 fluorescence was quantified using 633
nm excitation, with a 660/20 nm bandpass filter. Importantly, the
nozzle size to smoothly accommodate the 22.5 μm double emulsion
droplets was 130 μM. The selection threshold was deliberately
set close to the level of background noise, to create relatively permissive
screening conditions and avoid missing moderately improved catalysts
in library screens.^{49 (link),53 (link)}

SDD‐AGE experiments were performed based on protocols published by French et al (2019 (link)) and Halfmann and Lindquist (2008 (link)). First, 2 µM purified TDP‐43‐MBP‐His₆ variants (WT, 5D, 12D and 12A) were set up in low binding tubes (Eppendorf) in 35 µl aggregation buffer (50 mM Tris pH 8.0, 250 mM NaCl, 5% glycerol, 5% sucrose, 150 mM imidazole pH 8.0) and supplemented with 1× protease inhibitor (Sigma). Samples were shaken for 30 min on a thermomixer at 1,000 rpm at RT (~22°C) and then incubated at RT for the indicated time period. 5 µl of each sample was collected and diluted in SDD‐AGE buffer (40 mM Tris–HCl pH 6.8, 5% glycerol, 0.5% SDS, 0.1% bromphenol‐blue) and analyzed by SDD‐AGE by horizontal 1.5% agarose gel electrophoresis (gel: 1.5% agarose in 20 mM Tris, 200 mM glycine and 0.1% SDS) in running buffer (60 mM Tris, 20 mM acetate, 200 mM glycine, 1 mM EDTA and 0.1% SDS) for ~6 h at 60 V. Detection of TDP‐43 monomers, oligomers and high‐molecular‐weight species was performed after overnight capillary transfer in TBS (50 mM Tris pH 7.6, 150 mM NaCl) to a nitrocellulose membrane and by standard Western Blot using rabbit anti TDP‐43 N‐term antibody (Proteintech, Cat. No.: 10782‐2‐AP).