Coomassie simplyblue safestain

Microtubules were polymerized from 40 μM unlabeled tubulin in BRB80 (80 mM PIPES, 1 mM EGTA, 1 mM MgCl₂), 4 mM MgCl₂, 1 mM Mg-GTP, and 5% DMSO at 37 °C for 20 min. Polymerized microtubules were cleared by centrifugation at 17,000× g at 21 °C for 30 min. and then diluted in 114 µL PBST (PBS containing 10 μM taxol). Polymerized microtubules were then incubated with 500 nM tau, wildtype FUS or FUS-P525L-GFP, respectively, in PBST for 20 min at room temperature. Samples were then centrifuged at 45,000× g and 21 °C for 20 min, after which the supernatant was separated from the microtubule-containing pellet. SDS loading buffer (Thermo Scientific™, Waltham, MA, USA), supplemented with 0.1 M DTT, was mixed 1:4 with each sample and boiled at 95 °C for 5 min. Samples were loaded onto a 4–12% Bis-Tris gel (Thermo Scientific™, Waltham, MA, USA),. The gel was run in 1× MOPS buffer (Thermo Scientific™, Waltham, MA, USA) at 180 V, 85 W, and 60 mA until all protein had passed to the end of the gel (at least one hour). Gels were then stained with Coomassie SimplyBlue SafeStain (Thermo Scientific™, Waltham, MA, USA) for one hour and destained in ddH2O until bands were clearly visible (at least one hour). Gels were then scanned with an Azure c600 Gel Imaging System (Azure Biosystems Inc., Dublin, CA, USA).

Recombinant proteins were prepared as described previously and stored at −80 °C. Reactions were performed in microcentrifuge tubes in final volumes of 20 μl at 30 °C. Reactions contained 40 mM Tris–HCl (pH 7.5), 50 mM NaCl, 0.5 mM Tris(2-carboxyethyl)phosphine, 5 mM MgCl₂, 5 mM ATP, 100 μM Ub, 50 nM E1 protein (UBE1), 1 μM E2 protein (UbcH5c), and 1 μM E3 protein (NEDD4 or ITCH). Reactions were prepared without E1 enzyme, and a time 0 sample was taken (5 μl). E1 enzyme was added to initiate the reaction, and various time points were taken and quenched by adding Laemmli sample buffer (Bio-Rad) with 10% 2-mercaptoethanol to the mixture and boiling for 5 min. Samples were then run on SDS-PAGE gels and stained with Coomassie SimplyBlue SafeStain (Thermo Fisher Scientific).

At several time-points, SARS-CoV-2-infected Vero cells were washed twice with 5 ml of PBS to remove cell culture medium and FCS. Cells were recovered in 1.5 ml of PBS. The virus was inactivated and the cells lysed by autoclaving the samples at 125°C for 40 min. Proteins were precipitated by adding cold trichloroacetic acid to a final concentration of 10% (w/v). After 5 min at 4°C, the precipitated material was recovered by centrifugation at 16,000 g for 10 min. The proteins in the resulting pellets were dissolved in 100 μL LDS 1X (Lithium dodecyl sulfate) sample buffer (Invitrogen) supplemented with 5% beta-mercaptoethanol (vol/vol). Samples were sonicated with a Hielscher UP50H disruptor for 20 s operating at 60% amplitude, alternating 0.25-s impulsions with incubation for 5 min at 99°C. A 20-µl aliquot of a 1/8 dilution in LDS 1X (Invitrogen) of each sample was loaded on a NuPAGE 4–12% Bis-Tris gel and subjected to short (5-min) SDS-PAGE migration. Proteins were stained for 5 min with Coomassie SimplyBlue SafeStain (Thermo Fisher Scientific) prior to in-gel trypsin proteolysis, as described in Hartmann et al. [17 (link)].

The proteins from 5 g of soil were extracted using the NoviPure Soil Protein Extraction Kit (Mo-Bio) as recommended by the supplier. After centrifugation, proteins from the 10-ml supernatant were precipitated by adding 2.5 ml trichloroacetic acid (50% w/v). Proteins were collected by centrifugation for 10 min at 6000×g. The resulting pellet was resuspended in 40 μL LDS 1X (Invitrogen) containing 5% beta-mercaptoethanol, sonicated for 5 min in an ultra sound bath and then heated to 99 °C for 5 min. Soluble proteins (25 μL per well) were subjected to SDS-PAGE gel electrophoresis on NuPAGE 4–12% Bis-Tris gel (Invitrogen) for 5 min at 200 V in MES/SDS 1X running buffer (Invitrogen). Proteins were stained for 15 min with Coomassie SimplyBlue SafeStain (Thermo Fisher Scientific), and then in-gel proteolyzed with trypsin gold (Promega) for 1 h at 50 °C, as recommended [22 (link)].

A volume of 60 μL of inactivated viral particles sample containing 0.614 μg/μL of proteins was diluted with 20 μL of LDS1X (Invitrogen). The sample was heated at 99 °C for 5 min. A volume of 25 μL (11.5 μg) of this sample was loaded onto a NuPAGE 4–12% Bis-Tris gel. Proteins were subjected to a short (5 min) SDS-PAGE migration. The polyacrylamide gel was then stained with Coomassie SimplyBlue SafeStain (Thermo Fisher) for 5 min and then destained overnight with distilled water. The proteins were excised from the gel as a single polyacrylamide small band, reduced with 25 mM of dithiothreithol (Sigma-Aldrich) and treated with iodoacetamide (Sigma-Aldrich), and then subjected to in-gel trypsin proteolysis with Trypsin Gold (Promega) at a ratio of enzyme to protein of 2% and in the presence of 0.01% ProteaseMAX surfactant (Promega) following the procedure described by Hartmann et al. (2014). The peptide fraction obtained (50 μL) was analysed directly by tandem mass spectrometry.

Kanamycin was purchased from Carl Roth (Karlsruhe, Germany). LB agar, LB medium, MagicMedia E. coli expression medium, Novex 4–12% Bis-Tris gradient gels, Coomassie SimplyBlue SafeStain, fetal bovine serum, phosphate buffered saline (PBS), penicillin and streptomycin were obtained from Life Technologies (Darmstadt, Germany). BugBuster protein extraction reagent, Benzonase endonuclease and MMP-2 enzyme were purchased from Merck Millipore (Darmstadt, Germany), and Proteinase Halt protease inhibitor cocktail, maleimide-fluorescein, and bicinchoninic acid (BCA) protein assay were obtained from Thermo Fisher Scientific (Schwerte, Germany). BioGel P6 was bought from Bio-Rad Laboratories GmbH (Munich, Germany). Diethylaminoethyl (DEAE) cellulose, Octyl-Sepharose 4 Fast Flow and camptothecin were bought from Sigma-Aldrich (Steinheim, Germany). Maleimide-6S-IDCC was purchased from Mivenion GmbH (Berlin, Germany). Dithiothreitol (DTT), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethylene diamine tetraacetic acid (EDTA), trifluoroacetic acid (TFA), sodium carbonate, sodium thiosulfate, silver nitrate and all other chemicals were purchased from Sigma-Aldrich (Steinheim, Germany).