The protein samples (20 ug/well) were treated with 300 mM DTT at 95°C for 10 min and electrophoresed on a 12% Bis-Tris NuPAGE precast gel (1 mm) using the NuPAGE 12% Bis-Tris Electrophoresis System (Invitrogen, UK) according to the manufacturer’s instructions. Proteins were fixed by incubating in 50% (v/v) methanol for one hour with agitation. After two washes with deionised water for 20 min each, the gel was incubated with oxidizing agent (1% (w/v) periodic acid in 3% (v/v) acetic acid) for 2 hours with shaking, washed with deionised water two times for 20 min each, and incubated with Schiff’s reagent (Sigma-Aldrich, USA) for two hours. After removing Schiff’s reagent, the gel was incubated with reducing agent (2.5% (w/v) sodium metabisulphite in 10 mM hydrochloric acid) overnight, washed three times with deionised water to stop the reaction. An identical NuPAGE gel with samples were stained with Coomassie Brilliant Blue R-250 dye solution (0.125% (w/v) of Coomassie Brilliant Blue R250 (Sigma, USA) in 40% (v/v) methanol and 10% (v/v) acetic acid) and destained until the background is clear. Both PAS- and Coomassie-stained gels were imaged on Gel Doc XR+ System (Bio-Rad, USA).
Coomassie brilliant blue r 250
Manufactured by Merck Group
Sourced in United States, Germany, China, United Kingdom, France, Macao
Coomassie Brilliant Blue R-250 is a protein staining dye used in biochemical and analytical applications. It is a blue dye that binds to proteins, allowing their visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Gelatin, by Merck Group (22 mentions)
Bovine serum albumin (bsa), by Merck Group (16 mentions)
Triton x 100, by Merck Group (15 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (13 mentions)
Acrylamide, by Merck Group (12 mentions)
Trypsin, by Merck Group (10 mentions)
Methanol, by Merck Group (9 mentions)
Precision plus protein standard, by Bio-Rad (8 mentions)
Ammonium persulfate, by Merck Group (8 mentions)
Acetic acid, by Merck Group (8 mentions)
β mercaptoethanol, by Merck Group (8 mentions)
Tris base, by Merck Group (7 mentions)
Bromophenol blue, by Merck Group (6 mentions)
Glycerol, by Merck Group (6 mentions)
Proteinase k, by Merck Group (5 mentions)
Glycine, by Merck Group (5 mentions)
Bis acrylamide, by Merck Group (5 mentions)
Methanol, by Thermo Fisher Scientific (5 mentions)
Iodoacetamide, by Merck Group (5 mentions)
Sodium carbonate, by Merck Group (5 mentions)
359 protocols using coomassie brilliant blue r 250
1
Visualizing Glycoproteins in Polyacrylamide Gels
2
In vitro Cin8 Kinase Assay
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In vitro phosphorylation assays were performed as previously described 18 (link), 20 . In brief, bacterially expressed Cin8 (590)-TEV-EGFP-6His variants were purified using standard nickel affinity chromatography, eluted with 300mM imidazole that was subsequently removed using Zeba Spin Desalting Columns 40K (Thermo Scientific). For a phosphorylation assay, equal concentrations of Cin8 variants were mixed with TAP-purified Clb2-Cdk1- Cks1 complex in kinase assay mixture [50mM HEPES, pH 7.4, 150mM NaCl, 5mM MgCl2, 8% glycerol, 0.2 mg/ml BSA, 500nM Cks1, and 500μM ATP (with added γ-32P-ATP (PerkinElmer)]. Reactions were stopped after 10 and 20 min with SDS-PAGE sample buffer and proteins were separated by SDS-PAGE. Gels were stained with Coomassie Brilliant Blue (CBB) R-250 (Sigma) and incorporation of 32P into the proteins was visualized by autoradiography.
3
SDS-PAGE Analysis of Extracts
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A 100 mg sample of each of the G1S, E-Sup, and G2S extracts was resuspended in 50 μL SDS sample buffer (0.125 mM Tris-HCl, 4% SDS, 10% sucrose, 0.01% bromophenol blue, and 10% 2-mercaptoethanol) and heated to 95 °C for 1 min. Proteins were separated in a NIHON EIDO system (NIHON EIDO, Tokyo, Japan) using 12% Tris-Tricine gels, alongside a protein ladder (Spectra Multicolor Broad Range Protein Ladder, Thermo Fisher Scientific K.K.) to estimate molecular weights. Proteins were stained with Coomassie Brilliant Blue (CBB R-250) and 0.01% Stains-All (Sigma Chemical Co., St Louis, MO).
4
Recombinant Protein Purification and Characterization
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Agarose, Micro BCA protein assay kit, Ni-NTA resin, and PageRuler prestained protein ladder were purchased from ThermoFisher Scientific (Rockford, IL, USA). Ammonium sulfate, chloramphenicol, complete Freund’s adjuvant, Coomassie brilliant blue (CBB) R-250, 3,5-dinitrosalicylic acid (DNS), ethylenediamine tetraacetic acid (EDTA), d -galactose, incomplete Freund’s adjuvant, isopropyl β-d -1-thiogalactopyranoside (IPTG), kanamycin, naphthoresorcinol, sodium dodecyl sulfate (SDS), and tris(2-carboxyethyl)phosphine (TCEP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Restriction enzymes and T4 ligase were obtained from Roche (Basel, Switzerland). The pET-30a vector for expressing C-terminal 6× His-tagged protein and Immobilon-P membrane were purchased from EMD Millipore Corporation (Temecula, CA, USA). A standard NAOS mixture (NA2–NA18) was provided by Dr. Sang-Hyeon Lee (Lee et al. 2008 (link)). An AOS mixture was prepared by mild acid hydrolysis of 1%[w/v] Agarose as previously described (Kwon et al. 2020 (link)). The standards (NA2, NA4, and NA6) were obtained from Carbosynth Ltd. (Berkshire, UK). E. coli BL21 (DE3) pLysS was obtained from Novagen (Madison, WI, USA).
5
Purification and Characterization of Bovine Trypsin
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Bovine serum albumin (BSA) and bovine pancreatic trypsin were procured from Sisco Research Laboratory (Mumbai, India). DEAE-cellulose, trypsin-Sepharose 4B, Sephadex G-50, N-α-benzoyl-DL-arginine-p-nitroanilide (BApNA), Bowman-Birk inhibitor from soybean (BBI), sorbitol, tricine, gelatin, and coomassie brilliant blue (CBB) R-250 were purchased from Sigma (St. Louis, MO). Immobilized pH gradient (IPG) strips (pH 4–7 linear, 11 cm), IPG buffer (pH 4–7 linear), dithiothreitol (DTT), and iodoacetamide (IDA) were procured from GE Healthcare Bio-Sciences AB (Uppsala, Sweden). Bicinchoninic acid (BCA) protein estimation kit was purchased from Thermo Scientific (USA). The protein molecular mass standards (range 3.5–57 and 4.6–100 kDa) from Puregene, Genetix, India were used. All other chemicals and reagents used were of analytical grade.
6
Cellulase Purification and Characterization
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The cellulolytic microorganism isolates were used to inoculate 100 mL liquid medium C(CMC-Na 10 g, peptone 10 g, NaCl 5 g and 1000 mL water, adjust the pH to 7.0) at 37 °C. After 30 hours of culture, the broth was separated by centrifugation (8,000 g, 10 min) at 4 °C and the supernatant was collected. The purity of the cellulases was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 10% acrylamide gel and 5% condensing gel in a Mini-Protein II electrophoresis unit (Bio-Rad). The gel was stained with 0.25% Coomassie brilliant blue R-250 (Aldrich, USA). Running and staining procedures were performed according to the supplier’s protocol34 (link). The protein concentration was determined by the Bradford method with bovine serum albumin as a standard34 (link).
7
Protein characterization by SDS-PAGE, IEF, and SEC
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The fermentation solution and purified HMG fusion proteins from different purification processes were analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with 8% acrylamide gel and 5% condensing gel in the Mini-Protein II electrophoresis unit (Bio-Rad, USA) and stained with 0.25% Coomassie brilliant blue R-250 (Aldrich, USA).
IEF was used to predict the isoelectric point (pI) of HMG. In a separate set of experiments, 2 µg of purified HMG, mG-CSF, HSA and a mixture of HSA, mG-CSF and HMG prepared in 20 mM PB (phosphate buffer, pH 7.4) were loaded and analyzed on a Pharmacia MultiphorII horizontal electrophoresis system (GE Healthcare, USA) using ampholine, pH 3.5–10 (GE Healthcare, USA).
These samples were also analyzed using size exclusion chromatography on a TSK-GEL G3000SW columns (7.5×300 mm) (Tosoh, Japan) at a flow rate of 0.6 ml/min in 20 mM sodium phosphate (pH 7.5) and 0.15 M NaCl. The absorbance was monitored at 280 nm.
IEF was used to predict the isoelectric point (pI) of HMG. In a separate set of experiments, 2 µg of purified HMG, mG-CSF, HSA and a mixture of HSA, mG-CSF and HMG prepared in 20 mM PB (phosphate buffer, pH 7.4) were loaded and analyzed on a Pharmacia MultiphorII horizontal electrophoresis system (GE Healthcare, USA) using ampholine, pH 3.5–10 (GE Healthcare, USA).
These samples were also analyzed using size exclusion chromatography on a TSK-GEL G3000SW columns (7.5×300 mm) (Tosoh, Japan) at a flow rate of 0.6 ml/min in 20 mM sodium phosphate (pH 7.5) and 0.15 M NaCl. The absorbance was monitored at 280 nm.
8
Cloning and Expression of TspGST Protein
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The TspGST gene (GenBank accession no. XM_003373603) was amplified by PCR using specific primers with BamHI and Hind III restriction enzyme sites (underlined) (forward: 5′-TAT AGG ATC C AT GAC CAA CAC GTC GAA GAA AGG-3′; reverse, 5′-GCC CAA GCT T TC ATT GAC TTT CAA TAG TCA CTG G-3′). The purified PCR product was cloned into the pMD19-T vector (Takara, Dalian, China), subsequently sub-cloned into the pQE-80 L (Novagen, La Jolla, CA, USA). The recombinant plasmid carrying the TspGST gene was transformed into Escherichia coli BL21 (DE3) (Novagen), and expressed under IPTG induction. The rTspGST was purified using Ni-NTA-Sefinose resin (Sangon Biotech, Shanghai, China). The concentration of the purified rTspGST was assayed as described previously [22 (link)], and identified by SDS-PAGE analysis [23 (link)]. The gel was stained with 0.25% Coomassie brilliant blue R-250 (Sigma-Aldrich), and subsequently decolorized.
9
Protein Quantification and Visualization
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Protein concentration was determined using Bradford reagent (Bio-Rad) and BSA standards for preparation of a standard curve. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method of Laemmli (1970) (link), standard gels and a protein standard (Fermentas). Gels were stained using Coomassie Brilliant Blue R-250 (Sigma).
10
Proteome Analysis of Plant-Fungus Interaction
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The changes at the proteome level in the plant–fungus interaction were studied using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For SDS-PAGE, 250 µg/µL protein samples were loaded and run using Mini-PROTEAN Tetra Cell apparatus at 90 V (Bio–Rad, Hercules, CA, USA) in 12% gels with a molecular weight marker (Bench MarkTM Protein ladder, Life Technologies, Bangalore, India). Gels were stained using a solution of 0.2% (w/v) Coomassie Brilliant Blue R250 (Sigma-Aldrich, St. Louis, MO, USA), methanol (Sigma-Aldrich, St. Louis, MO, USA) 40% (v/v) and acetic acid (Sigma-Aldrich, St. Louis, MO, USA) 10% (v/v) for 90 min and destained with a solution of methanol 40% (v/v) and acetic acid 10% (v/v) for 90 min and later stored in acetic acid 5% (v/v) at room temperature.
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