To visualize the proteins present, CFS samples were analyzed via SDS-PAGE. For this, 5 mL of CFS was concentrated to ∼500 μL using Amicon Ultra 0.5 Centrifugal Filter Units with 10 kDa nominal molecular weight cut-off (Merck, Darmstadt, Germany). Approximately equal total amounts of protein were loaded onto an SDS-PAGE gel, dyed using InstantBlue Stain (Sigma-Aldrich, MO, United States).
Instant blue stain
Manufactured by Merck Group
Sourced in United Kingdom
Instant Blue stain is a laboratory reagent used for the rapid and sensitive detection and quantification of proteins in various applications, including SDS-PAGE, Western blotting, and dot blotting. It provides a quick and reliable way to visualize protein bands or spots on membranes or gels.
Automatically generated - may contain errors
Lab products found in correlation
Amicon ultra 0.5 centrifugal filter unit, by Merck Group (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Nupage mes sds running buffer, by Thermo Fisher Scientific (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nupage novex 3 8 tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor tablet, by Roche (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Pancreatin, by Merck Group (1 mentions)
Nupage mops buffer, by Thermo Fisher Scientific (1 mentions)
Brilliant blue r concentrate, by Merck Group (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Typhoon trio imager, by GE Healthcare (1 mentions)
G box system, by Syngene (1 mentions)
Anti dnp, by Merck Group (1 mentions)
Dinitrophenylhydrazine dnph, by Merck Group (1 mentions)
Genesys software, by Syngene (1 mentions)
5 protocols using instant blue stain
1
SDS-PAGE Protein Visualization
2
Native and Denatured Protein Electrophoresis
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Proteins were subjected to native gel electrophoresis using NuPAGE Novex 3–8% Tris-acetate gels and Novex Tris-glycine native buffer (Life Technologies). Denaturing gel electrophoresis was performed using NuPAGE Novex 4–12% Bis-Tris gels and NuPAGE Mes SDS running buffer (Life Technologies). Where specified, samples were reduced by treatment with 5% (v/v) β-mercaptoethanol. Gels were stained using Instant Blue stain (Sigma-Aldrich).
For Western blot analysis heparinised human plasma was supplemented with cOmplete protease inhibitor cocktail according to the manufacturer's instructions and diluted to 2 mg/ml in PBS/Az. The plasma was supplemented with 0 or 320 μM NaOCl and left to incubate at ambient room temperature overnight. Following reduction using 5% (v/v) β-mercaptoethanol, the proteins were separated using a NuPAGE Novex 4–12% Bis-Tris gel and transferred to PVDF membrane using an iblot 2 (Life Technologies) according to the manufacturer's instructions. After blocking overnight at 4 °C using 5% (w/v) skim milk powder in PBS, the membrane was incubated with polyclonal goat anti-fibrinogen antibody (Life Technologies; diluted 1:2000 in blocking solution) and then incubated with a superclonal anti-goat IgG-HRP conjugate (Life technologies; diluted 1:5000 in blocking solution). Blots were imaged by enhanced chemiluminescence using a Chemidoc touch imaging system (Bio-Rad).
For Western blot analysis heparinised human plasma was supplemented with cOmplete protease inhibitor cocktail according to the manufacturer's instructions and diluted to 2 mg/ml in PBS/Az. The plasma was supplemented with 0 or 320 μM NaOCl and left to incubate at ambient room temperature overnight. Following reduction using 5% (v/v) β-mercaptoethanol, the proteins were separated using a NuPAGE Novex 4–12% Bis-Tris gel and transferred to PVDF membrane using an iblot 2 (Life Technologies) according to the manufacturer's instructions. After blocking overnight at 4 °C using 5% (w/v) skim milk powder in PBS, the membrane was incubated with polyclonal goat anti-fibrinogen antibody (Life Technologies; diluted 1:2000 in blocking solution) and then incubated with a superclonal anti-goat IgG-HRP conjugate (Life technologies; diluted 1:5000 in blocking solution). Blots were imaged by enhanced chemiluminescence using a Chemidoc touch imaging system (Bio-Rad).
3
Producing Infectious CPMV and eVLPs in Plants
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pBinPS1NT and pBinPS2NT were used for the production of infectious CPMV in plants28 (link) while pEAQexpress-VP60-24 K was used for the expression of eVLPs.4 (link),29 Complete® Protease inhibitor tablets were bought from Roche (UK). 12% (w/v) NuPAGE polyacrylamide bis–tris gels, NuPAGE MOPS buffer, NuPAGE LDS Sample Buffer were obtained from Invitrogen (UK). Brilliant Blue R Concentrate and InstantBlue stain were purchased from Sigma (UK) and Expedon (UK), respectively. Amicon centrifugal filters (100 kDa MWCO) and Micro Float-A-Lyzer dialysis tube (100 kDa MWCO) were purchased from Millipore and Spectrum Laboratories, respectively. Bovine Serum Albumin (BSA, heat shock fraction), pepsin (≥400 units per mg protein) from porcine gastric mucosa and pancreatin (≥3 × USP specifications) from porcine pancreas were obtained from Sigma-Aldrich (UK). Three full-length gastrointestinal tracts were procured from freshly killed pigs at a local abattoir (H. G. Blake Costessey Ltd, Norwich, UK). The animals were processed under standard UK legislation for food-producing animals, the gut extracted within less than an hour of slaughter and transported intact to the laboratory on ice.
A summary of the composition of the media used in this study is presented inTable 1 .
A summary of the composition of the media used in this study is presented in
4
Protein Characterization by Gel Electrophoresis
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Proteins were subjected to native gel electrophoresis using NuPAGE Novex 3 to 8% Tris-acetate gels and Novex Tris-glycine native buffers (Life Technologies). Denaturing gel electrophoresis was performed using NuPAGE Novex 4 to 12% Bis-Tris gels and NuPAGE Mes SDS running buffer. Where specified, samples were reduced by treatment with β-mercaptoethanol. Gels were stained using Instant Blue stain (Sigma-Aldrich). In experiments involving Hilyte-labeled Aβ1–42, the migration of the fluorescently labeled peptide was determined using a Typhoon Trio imager (GE Healthcare).
For Western blot analysis, proteins were subjected to electrophoresis as described above and transferred to nitrocellulose or polyvinylidene difluoride (PVDF) membrane. After blocking overnight at 4 °C in skim milk solution [5% (wt/vol) skim milk powder in PBS], the membranes were incubated with the relevant antibodies/streptavidin conjugates diluted in skim milk solution (1 h at 37 °C). Blots were imaged with an Amersham 600 imager (GE Healthcare) using enhanced chemiluminescence. PZP was detected using an affinity-purified polyclonal antibody (GeneTex), α2M was detected using monoclonal antibody 2N1/10 (Bio-Rad) or a polyclonal antibody (Dako), and Aβ1–42 was detected using monoclonal antibody W02 supernatant.
For Western blot analysis, proteins were subjected to electrophoresis as described above and transferred to nitrocellulose or polyvinylidene difluoride (PVDF) membrane. After blocking overnight at 4 °C in skim milk solution [5% (wt/vol) skim milk powder in PBS], the membranes were incubated with the relevant antibodies/streptavidin conjugates diluted in skim milk solution (1 h at 37 °C). Blots were imaged with an Amersham 600 imager (GE Healthcare) using enhanced chemiluminescence. PZP was detected using an affinity-purified polyclonal antibody (GeneTex), α2M was detected using monoclonal antibody 2N1/10 (Bio-Rad) or a polyclonal antibody (Dako), and Aβ1–42 was detected using monoclonal antibody W02 supernatant.
5
SDS-PAGE and Oxyblotting for Protein Carbonylation Analysis
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Aliquots of the samples (10 µL; ~ 75 µg protein) were resolved by SDS-PAGE with a 12% resolving gel (Sambrook and Russell 2006 ) and then either stained with InstantBlue stain (Sigma-Aldrich, UK) or transferred onto PVDF membrane for oxyblotting as described previously (Shacter 2000 (link)). After washing, the membrane was acidified with 2 N HCl and labelled with 10 mM dinitrophenylhydrazine (DNPH) (Sigma-Aldrich, UK) for 5 min. After further washing and blocking the membrane was incubated in blocking buffer containing monoclonal primary antibodies rabbit anti-dinitrophenylhydrazone (anti-DNP) (D9656, Sigma-Aldrich, UK) at a working dilution of 1:1000 overnight at 4 °C. The secondary antibody was HRP-linked goat anti-mouse (6154, Sigma-Aldrich, UK) antibody (working dilution 1:1000) for 2 h at room temperature. The membrane was washed again as described above and HRP-linked anti-mouse was detected using enhanced chemiluminescence (ECL kit 34078, Thermo Fisher Scientific, Hemel Hempstead, UK) according to the manufacturer’s instructions. The membrane was scanned using a G:BOX system (Syngene, Cambridge, UK) running the GeneSys software (Syngene, Cambridge, UK).
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