Coomassie r 250

To determine the primary structure of proteins in the plasma exosomes of HFs and BCPs, proteins were separated using 10–20% SDS-disk electrophoresis and stained with Coomassie R-250 (Sigma, St. Louis, MO, USA). Fragments of polyacrylamide gel containing the studied proteins were washed from Coomassie R-250 and SDS and subjected to trypsinolysis as described previously [28 (link)]. Peptide fragments of proteins were extracted from the gel, concentrated, and desalted on C18 ZipTips microcolumns (Milipore, Burlington, MA, USA). The mixture of peptides was eluted from the microcolumn onto the instrument plate target with a saturated matrix solution.
The acquisition and registration of mass spectra was carried out on a time-of-flight tandem mass spectrometer MALDI-TOF autoflex speed series LIFT (Bruker Daltonics, Bremen, Germany) at the Mass Spectrometry Research Center of the Siberian Branch of the Russian Academy of Sciences. Protein identification was performed by searching for appropriate candidates in the annotated NCBI and SwissProt databases using the Mascot program (Matrix Science Ltd., London, UK, www.matrixscience.com/search_form_select.html accessed on 25 February 2022) as described in [28 (link)].

The spots corresponding to the Hsp70 were manually extracted from the Coomassie R-250 (Sigma-Aldrich) stained 2DE gels of the L. prolificans total protein extract. Then they were electroeluted with the 422 Electoeluter (Bio-Rad) system, in accordance with manufacturer’s instructions. Finally, a 1DE was made with the electroeluted samples, using 12% gels.

Cells were incubated with GlaB and C22 chemical compounds for 24 h under normoxia and hypoxia and then media were collected and centrifuged at 300× g for 5 min. Total protein amount was analysed with a Micro BCA Protein Assay Reagent kit (Thermo Fisher Scientific, Cleveland, OH, USA) and 10 μg of diluted medium sample was resolved by 1% gelatin from porcine skin–10% acrylamide gels and incubated overnight at 37 °C with agitation in developing buffer. The next day, gels were stained with Coomassie R-250 (Sigma Aldrich, Saint Louis, MO, USA) for the detection of enzymatic activity through colorimetric staining, and images were acquired with a ChemiDoc™ MP System and quantified with ImageJ software 1.53K (http://imagej.nih.gov/ij/docs/index.html) (accessed on 5 February 2024).

Electrophoretic analysis (EA) of proteins in gradient 5–15% of polyacrylamide gel electrophoresis (PAGE) in the presence 10% β-mercaptoethanol (Figure 6A) was performed according to standard procedure [52 ]. Staining of PAGE was performed while using Coomassie R-250 (Sigma-Aldrich, St. Louis, MO, USA).

Clonogenic assays were performed in 10 cm tissue culture dishes (for RPE-1 cells, U2OS cells and derived clones). Cells (1000 per dish) were seeded in complete media (10 mL) and allowed to attach overnight, then treated with cisplatin or cisplatin plus inhibitor for the times stated. Following treatments, cells were allowed to grow and form colonies for 10 days. Colonies formed were stained with Coomassie R250 (Sigma) and counted on a COLCount Colony Counter (Oxford Optronix). All experiments represent the mean (±SEM) of at least three biological repeats of duplicate dishes/flasks for each treatment.

Cytoplasmic extracts from serum-starved (60 min) OCI/AML3 cells (about 4 mg) were loaded on a HiTrap SP column (GE Healthcare, Chicago, IL, USA). Then, the resulting flow-through was directly loaded on a HiTrap Benzamidine column (GE Healthcare, Chicago, IL, USA), which was eluted with 50 mM glycine, pH 3. Experiments were carried out on an AKTA Prime FPLC system (GE Healthcare, Chicago, IL, USA). Resulting elution fractions were tested with the above-mentioned in vitro proteolysis assay, resolved in SDS-PAGE, and stained with Coomassie R250 (Sigma-Aldrich, St. Louis, MO, USA). Positive fractions were then pooled, and subjected to proteomic analysis for protease identification.

The purified OsNifH^Ht protein was quantified by the densitometric analysis of Coomassie-stained gels using ImageJ^{45 (link)} compared to a standard of yeast ScNifB^Mi protein as shown in Supplementary Fig. 4. N-terminal amino acid sequences were determined by Edman degradation (Centro de Investigaciones Biológicas, Madrid, Spain). OsNifH^Ht protein (~250 pmol) was separated by SDS-PAGE, transferred to a Sequi-Blot PVDF membrane with a 0.2 µm pore size (Thermo Fisher Scientific) in 50 mM borate buffer (pH 9.0), stained with freshly prepared Coomassie R-250 (Sigma-Aldrich, St Louis, MO, USA) (0.1% in 40% methanol and 10% acetic acid), and then destained using 50% methanol. OsNifH^Ht protein (~60 pmol) was separated by SDS-PAGE followed by Coomassie staining and destaining as described above. Protein excised from the gel was analyzed by mass spectrometry at the Universidad Complutense de Madrid, Spain.