HFIP-treated Aβ1-42 stored at -80°C in DMSO was oligomerised by dilution and vortexing in PBS followed by incubation overnight at 4°C [25 (link)]. Oligomer formation was confirmed by native tricine-SDS-polyacrylamide gel electrophoresis. Briefly, 2 μg oligomeric Aβ (oAβ) was resuspended in nondenaturing sample buffer (62.5 mM Tris-base, 25% glycerol, and 1% (w/v) Coomassie Blue R-250) and loaded onto a 10% acrylamide : bis-acrylamide gel and separated by electrophoresis alongside molecular weight markers. Gels were incubated with Coomassie stain (60 mg/l Coomassie Blue R-250 and 10% v/v acetic acid, both from Sigma-Aldrich, UK). Following 24 h destaining in 10% v/v acetic acid and 50% v/v methanol (Sigma-Aldrich, UK), gels were imaged using a ChemiDoc MP Imaging System (Bio-Rad Ltd., UK). Oligomeric Aβ migrated at approximately 35 kDa, indicating the presence of hexamers/heptamers (Supplementary Figure 1 ).
Coomassie stain
Manufactured by Merck Group
Sourced in United Kingdom, Germany
Coomassie stain is a protein-binding dye used in biochemistry and molecular biology laboratories to detect and quantify proteins in various applications, such as gel electrophoresis and Western blotting. It provides a simple, sensitive, and reliable method for visualizing and analyzing protein samples.
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Lab products found in correlation
Coomassie blue r 250, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Vinculin hvin 1, by Merck Group (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
6 protocols using coomassie stain
1
Amyloid-β oligomer formation protocol
2
Quantitative Western Blotting of Dystrophin and Utrophin
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Samples for dystrophin and utrophin westerns were prepared, and protein levels quantified as previously described (43 (link)). Briefly, homogenised samples were run on 3–8% Tris-acetate gels (Thermo Fisher Scientific), blotted onto PVDF membrane (Millipore), and probed with DYS1 (Novocastra, dystrophin) or MANCHO antibody (KED laboratory—Oxford, utrophin). Blots were visualised using IRDye 800CW goat-anti mouse IgG (LI-COR) on the Odyssey imaging system. For dystrophin, vinculin (hVIN-1; Sigma-Aldrich) was used as loading control, and for utrophin, the samples were quantified against total protein using Coomassie stain (Sigma-Aldrich).
3
Immunoblot Analysis of Fusion Proteins
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Immunoblots were analyzed as described in Gardezi et al. (2016 (link)). Immunoblots were imaged with the ChemiDoc (Bio-Rad, Hercules, CA, USA) with a broad range of exposure times. For each experiment, protein band intensities were quantified by densitometry from a common blot at a single exposure selected for clear bands without saturation. Background counts were subtracted using an automated rolling disk subtraction. Protein band intensities were normalized to a single control condition for each experiment. For experiments comparing different fusion proteins, intensities were normalized to the C2 fusion protein intensity. For peptide experiments, intensities were normalize to the control peptide condition as described previously (Gardezi et al., 2016 (link)).
Fusion protein concentrations were visualized using Coomassie stain (Sigma-Aldrich) and imaged with the ChemiDoc (Bio-Rad) Coomassie stain protein gel function. Concentrations were quantified by densitometry and background was subtracted using an automated rolling disk subtraction. Fusion protein concentration was used as a loading control so that SV2 (ISV2) and STG (ISTG) protein intensities were normalized to the fusion protein concentration (IFP) from the same lane, hence %SV-PD was calculated as ISV2/IFP or ISTG/IFP respectively.
Fusion protein concentrations were visualized using Coomassie stain (Sigma-Aldrich) and imaged with the ChemiDoc (Bio-Rad) Coomassie stain protein gel function. Concentrations were quantified by densitometry and background was subtracted using an automated rolling disk subtraction. Fusion protein concentration was used as a loading control so that SV2 (ISV2) and STG (ISTG) protein intensities were normalized to the fusion protein concentration (IFP) from the same lane, hence %SV-PD was calculated as ISV2/IFP or ISTG/IFP respectively.
4
Coomassie Brilliant Blue Protein Staining
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Following electrophoresis, the gel was soaked in fixing buffer consisting of 40% (v/v) ethanol and 10% (v/v) acetic acid for 1 h. After fixation, the gel was washed for 1 h with deionized water and incubated for 2 h in Coomassie stain (Sigma) with continuous agitation. The gel was destained with deionized water.
5
Cathepsin Zymography for Enzyme Activity
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Cathepsin zymography was performed as previously described41 (link). Briefly, 6X non reducing loading buffer (0.05% bromophenol blue, 0.5 M Tris HCl, 50% glycerol, 10% SDS) was added to all samples prior to loading. Samples were then resolved by 12.5% SDS-PAGE containing 0.2% gelatin at 4 °C (30 mA/gel, 0.75 mm thick minigel). The gels were carefully removed and enzymes renatured 3X for 10 minutes in renaturing buffer (65 mM Tris buffer, pH 7.4 with 20% glycerol). Afterwards, gels were incubated in activity buffer (0.1 M phosphate buffer, 1 mM EDTA and freshly added 2 mM DTT pH 6.0) for 30 minutes at room temperature and then changed to a fresh activity buffer and incubated at 37 °C for 72 hours. Gels were then washed twice with distilled water and then stained for 30 minutes in coomassie stain (0.5% coomassie (Sigma, Steinheim, Germany) in 30% ethanol and 10% acetic acid). Gels were then destained in destaining solution(30% ethanol and 10% acetic acid) until clear bands appeared. To also detect secreted CatK by western blot, SDS page was carried out as outlined above without gelatin substrate polymerized in the resolving gel. Protein sample was transferred to a PVDF membrane and probed with the CatK antibody (dilution 1:500, SantaCruz biotechnology, Heidelberg, Germany).
6
Scaffolds Staining Optimization Protocol
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Cell culture scaffolds (collagen, fibrin and non-coated, n=3/group) were stained with a coomassie stain (Sigma-Aldrich) for 30 min, and then rinsed three times with PBS and de-ionized (DI) water, until no traces of dye were detected in non-coated controls. The light microscope images were then taken, and the results were compared.
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