For growth experiments in different carbon source-supplemented media, B-medium was supplemented with 0.5% (w/v) AX, 0.5% (w/v) crystalline chitin (CHI), 0.5% (w/v) XG, 0.2% (w/v) heat-treated C.vulgaris biomass (C.v.) and 0.2% (w/v) C.vulgaris AIS (C.v. AIS). B-medium without any carbon source (NS) was used as negative control. Chitin from shrimp shells was purchased from Sigma-Aldrich (Saint Louis, USA). All polysaccharides were sterilized and supplied to B-medium (pH 6.5). Filtrates from culture media were prepared and assayed according to the procedure previously described. Alternatively, the filtrates were separated in a TGX™ Precast Protein Gel [4–15% (w/v) polyacrylamide] (Biorad, CA, USA) using Precision Plus Protein™ Dual Color Standards (Biorad, CA, USA) as molecular weight marker and then stained by silver nitrate. Proteolytic assay was performed by incubating 1 µg BSA with 20 µL of C.v.-filtrate for 16 h at 28 °C. BSA was purchased from Sigma-Aldrich (St. Louis, USA). The reaction was separated in 10% of Laemmli gel using Precision Plus Protein™ Dual Color Standards (Biorad, CA, USA) as molecular weight marker and then stained by silver nitrate.
Precision plus protein dual color standard
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Japan, Italy
Precision Plus Protein Dual Color Standards is a pre-stained protein molecular weight standard used for protein separation and analysis. It contains a mixture of ten recombinant proteins with molecular weights ranging from 10 to 250 kDa, which are stained with two different colored dyes. The standard is designed for use in SDS-PAGE, Western blotting, and other electrophoretic applications to determine the molecular weights of unknown protein samples.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (11 mentions)
Laemmli sample buffer, by Bio-Rad (9 mentions)
Mini protean tetra cell, by Bio-Rad (9 mentions)
Mini protean tgx precast gel, by Bio-Rad (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Immobilon p, by Merck Group (6 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (5 mentions)
Tris glycine sds buffer, by Bio-Rad (5 mentions)
Bradford assay, by Bio-Rad (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (4 mentions)
2 mercaptoethanol, by Bio-Rad (4 mentions)
Image lab software, by Bio-Rad (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
Laemmli buffer, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Immun blot pvdf membrane, by Bio-Rad (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
237 protocols using precision plus protein dual color standard
1
Growth Media Carbon Source Experiments
2
Purification of Recombinant Proteins in E. coli
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Individual clones of transformed Escherichia coli M15 cells were grown in LB medium with ampicillin until they reached an optical density at 600 nm of 0.6, and then the recombinant proteins were induced by the addition of isopropyl-β-D-1-thiogalactopyranoside to a final concentration of 1 mM (IPTG, Promega, Madison, WI, USA) for 3 h. The recombinant proteins were then purified by Ni-NTA Spin Column System (Qiagen, Hilden, Germany). The protein concentrations were determined using a Bradford assay (Bio-Rad, Hercules, CA, USA). Recombinant protein samples were analyzed by SDS-PAGE electrophoresis using nUView Precast Gels Tris-Glycine NB 4–20% (NuSep Inc., Germantown, MD, USA) and stained with Coomassie blue. Precision Plus ProteinTM Dual Color Standards (BIO-RAD, Hercules, CA, USA) were used as the molecular weight marker.
3
SDS-PAGE Protein Separation and Visualization
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SDS-PAGE was performed using Bis-Tris 4–12% gradient gels (Invitrogen) in a MES running system. Each sample contained 6 x protein loading dye containing DTT and was boiled for 5 min at 95 °C. Precision Plus ProteinTM Dual Color Standards (Biorad) or the Triple-color Protein Ladder One (Product No. 09547-74, Nacalai Tesque) were used as the molecular weight standards. The gel was run for 60 min at 140 V and stained with Coomassie blue (0.1% w/v Brilliant Blue R, Sigma), 40% methanol, 10% acetic acid in water and de-stained in 40% methanol, 10% acetic acid in water.
4
Leaf Protein Extraction and SDS-PAGE
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Total leaf proteins were extracted by incubating ground material in Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% β-mercaptoethanol) for 30 min at RT. After centrifugation for 10 min at 13,000× g, the supernatants were collected and the protein concentration was determined using a bicinchoninic acid kit (BCA 1, Sigma, Sigma-Aldrich, St. Louis, MO, USA). The protein samples were separated on 12% SDS-PAGE according to Okadjima et al. [40 (link)] using vertical electrophoresis unit SE 250 Mighty Small (Hoefer, San Francisco, CA, USA). Following SDS-PAGE, the gels were stained with 0.006% (w/v) Coomassie Brilliant Blue (CBB) R-250 in 50% (v/v) CH3OH, 10% (v/v) CH3COOH), or blotted on membrane. As molecular mass standards, prestained Precision Plus ProteinTM Dual Color Standards (Bio-Rad, Hercules, CA, USA) were used.
5
Characterization of PEGylated Zein Nanoparticles
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Yellow zein, Nile red, and all other chemicals that are not specifically mentioned below were purchased from Sigma-Aldrich (Poole, UK). Methoxy PEG succinimidyl carboxymethyl ester with a MW of 5 kDa (mPEG-SCM-5000) and 10 kDa (mPEG-SCM-10K) were obtained from JenKem Technology (Plano, TX, USA). Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 cell culture media, L-glutamine, fetal bovine serum (FBS), penicillin-streptomycin, and TrypLE® Express came from Life Technologies (Paisley, UK). 4–20% Mini-PROTEAN® TGXTM gels, Precision Plus ProteinTM Dual Color Standards, 10× TGS buffer, 2× Laemmli sample buffer, and Silver Stain PlusTM Kit were purchased from Bio-Rad (Watford, UK). Vectashield® mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) came from Vector Laboratories (Peterborough, UK). Bioware® B16-F10-luc-G5 mouse melanoma cells were purchased from Caliper Life Sciences (Hopkinton, MA, USA).
6
SARS-CoV-2 N Protein Detection by Western Blot
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Vero E6 cells were infected at an MOI of 0.1, harvested 16 h p.i., and lysed in RIPA buffer (Thermo Scientific, Illinois, USA). Equal volumes of cell lysate and 2× sample buffer (4% sodium dodecyl sulfate [SDS], 0.25 M Tris-HCl [pH 6.8], 2% 2-mercaptoethanol, 20% glycerol, and 0.01% bromophenol blue) were mixed and boiled at 100 °C for 10 min. The proteins in the sample were separated by 5%-20% SDS-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was stained with a rabbit antibody against the SARS-CoV-2 N protein (Gene Tex, CA, USA; 1:500 dilution) as the primary antibody, followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (102 PU, American Qualex, CA, USA). The protein bands were detected by immersing the membrane in 3,3′-diaminobenzidine tetrahydrochloride solution. For molecular weight estimation on the WB, Precision Plus ProteinTM Dual Color Standards (Bio-Rad, California, USA) were used. ImageJ software was used to measure the intensity of protein bands.
7
SDS-PAGE Protein Profiling of FBPIs
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SDS-PAGE was performed based on the method described by Laemmli [27 (link)] to determine the protein patterns of the FBPIs. The protein suspensions were dissolved in 5% w/v SDS solution and heated at 95 °C for 1 h followed by centrifugation at 7000× g for 10 min at 25 °C. The supernatants were mixed with a Laemmli sample buffer containing 2% SDS, 10% glycerol, and 0.05% bromophenol blue in 0.5 M Tris–HCl (pH 6.8) under non-reducing and reducing (+5% β-mercaptoethanol) conditions. Finally, the proteins (15 µg) were loaded onto the polyacrylamide gel (4% stacking gel; 12% running gel). Electrophoresis was performed at a constant current of 15 mA/gel until completion, followed by staining with Coomassie Brilliant Blue R-250 and destaining. Precision Plus ProteinTM dual color standards (Bio-Rad) were run concurrently for molecular weight estimations.
8
Purification and Characterization of Endoxylanase
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The estimated molecular mass of the enzyme and its purity were evaluated by SDS-PAGE in 12% acrylamide gels [20 (link)] using Precision Plus Protein TM Dual Color Standards (Bio-Rad) and staining with Coomassie Brilliant Blue R-250. The accurate protein molecular mass was determined by MALDI-TOF in an Autoflex III (Bruker Daltonics).
The determination of optimum pH and temperature together with thermal and pH stabilities for 72 h were performed as described elsewhere [15 (link)].
Enzyme kinetics were determined against beechwood xylan, measuring endoxylanase activity with increasing concentrations of substrate from 1 to 45 g/L and adjusting the experimental data by least-squares to the Lineweaver–Burk linear equation of the Michaelis–Menten model.
The determination of optimum pH and temperature together with thermal and pH stabilities for 72 h were performed as described elsewhere [15 (link)].
Enzyme kinetics were determined against beechwood xylan, measuring endoxylanase activity with increasing concentrations of substrate from 1 to 45 g/L and adjusting the experimental data by least-squares to the Lineweaver–Burk linear equation of the Michaelis–Menten model.
9
SDS-PAGE Protein Separation and Visualization
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The protein samples were diluted with Laemmli sample buffer containing 2-mercaptoethanol (5%, v/v) as a reducing agent at the volume ratio of 1:1. The samples (5 µg protein) were reduced at 90 °C for 5 min and loaded onto a 4–20% Mini-PROTEAN® TGXTM gel (Bio-Rad, Watford, UK). The gel was run with Tris/glycine/SDS buffer at 120 V for 1 h with Precision Plus ProteinTM Dual Color Standards (Bio-Rad, Watford, UK) as a molecular standard. The gel was stained with a Silver Stain PlusTM Kit (Bio-Rad, Watford, UK), as described in the kit’s instruction manual.
10
SDS-PAGE Fractionation of Synovial Fluid Proteins
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Synovial fluid samples were obtained from individual donors (n = 8), each separately labeled with a different number. Before fractionation, samples were diluted to 3% with sterile PBS. Aggregates formed as above were pelleted at 13,000 × g, 3 min, washed, and resuspended in 1 ml PBS by repeated vortexing and pipetting. Laemmli sample buffer with β-mercaptoethanol was added (1:1), samples heated at 70°C for 10 min, placed on ice, and fractionated (105 V, ∼70 min) on an SDS-polyacrylamide gel (7.5%), using the discontinuous buffer system of Laemmli (1970) (link). MW standards (10–250 kDa; Precision Plus ProteinTM Dual Color Standards, Bio-Rad, Hercules, CA, United States) were run on the same gel, as were serum, Fg (Alfa Aesar), and Alb (Thermo Fisher Scientific).
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