Cells were collected in RIPA buffer (10 mM Tris-HCl (pH 8.0), 1% (w/v) NP40, 0.1% (w/v) sodium deoxycholate (Wako), 0.1% (w/v) SDS (Wako), 0.15 M NaCl (Wako), 1 mM EDTA, 10 mM NaF (Wako), 1.5 mM Na3VO4 (Wako), and cOmplete™ Protease Inhibitor Cocktail (Roche)). Protein concentrations were titered using the BCA protein assay kit (Thermo Fisher Scientific). Collected protein lysate was mixed with SDS-PAGE loading buffer (0.15 M Tris-HCl, 6% (w/v) SDS, 0.003% (w/v) bromophenol blue (Wako), 30% (w/v) glycerol (Wako), and 15% (w/v) β-mercaptoethanol (Wako)) and then boiled at 95 °C for 5 min, followed by SDS-PAGE and immunoblotting. Antibodies used for immunoblotting were as follows: anti-milk (rabbit, 1:1000; Nordic-MUbio, Susteren, the Netherlands), anti-PyMT (rat, 1:500; Santa Cruz), anti-mCherry (rabbit, 1:500; Abcam), anti-histone H3 (rabbit, 1:2500; Cell Signaling Technology, MA, USA), and anti-α-tubulin (mouse, 1:5000; Calbiochem).
Bromophenol blue
Manufactured by Fujifilm
Sourced in Japan
Bromophenol blue is a pH indicator dye commonly used in laboratory settings. It is a chemical compound that undergoes a color change in response to changes in pH, making it useful for various analytical and experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Sodium dodecyl sulfate (sds), by Fujifilm (5 mentions)
Glycerol, by Fujifilm (4 mentions)
Glycine, by Fujifilm (2 mentions)
E pagel, by ATTO Corporation (2 mentions)
2 mercaptoethanol, by Fujifilm (2 mentions)
Sodium chloride (nacl), by Fujifilm (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol 2 me, by Merck Group (2 mentions)
Sodium deoxycholate, by Fujifilm (1 mentions)
Na3vo4, by Fujifilm (1 mentions)
β mercaptoethanol, by Fujifilm (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Fujifilm (1 mentions)
Anti mcherry, by Abcam (1 mentions)
Anti histone h3, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Methyl red, by Fujifilm (1 mentions)
Bromocresolgreen, by Fujifilm (1 mentions)
Bromocresol purple, by Fujifilm (1 mentions)
Cresol red, by Fujifilm (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
11 protocols using bromophenol blue
1
Protein Extraction and Immunoblotting
2
Fabrication of Dye-Based Colorimetric Paper
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Bromophenol blue, methyl red, bromocresol
green, disperse orange
3, bromocresol purple, fluorescein, and cresol red were purchased
from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). EA, DET,
and TMA were purchased from Alfa Aesar Co. (Great Britain). All other
reagents were of analytical grade and used as received. For the fabrication
of the DCP, Whatman chromatography paper grade 1 (200 × 200 mm,
pure cellulose paper) was obtained from GE Healthcare (China). The
designed DCPs were printed on a sheet of Whatman grade #1 using a
wax printer (ColoQube 8570DN, Xerox, USA) followed by heating at 120
°C for 2 min on a drying machine. Wax ink was used for the paper
hydrophobization and an insulation agent in this work.
The DCP
consisted of circles with an inside diameter of 5 mm and a border
of 2 mm (after melting wax ink). To prepare the DCP, the circles were
filled with 10 μL of 0.10 mM dye solutions and dried for 1 h
at room temperature. The colors of the DCP were recorded with a smartphone.
The spots on DCP with different dyes were assigned as shown inFigure 7 .
green, disperse orange
3, bromocresol purple, fluorescein, and cresol red were purchased
from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). EA, DET,
and TMA were purchased from Alfa Aesar Co. (Great Britain). All other
reagents were of analytical grade and used as received. For the fabrication
of the DCP, Whatman chromatography paper grade 1 (200 × 200 mm,
pure cellulose paper) was obtained from GE Healthcare (China). The
designed DCPs were printed on a sheet of Whatman grade #1 using a
wax printer (ColoQube 8570DN, Xerox, USA) followed by heating at 120
°C for 2 min on a drying machine. Wax ink was used for the paper
hydrophobization and an insulation agent in this work.
The DCP
consisted of circles with an inside diameter of 5 mm and a border
of 2 mm (after melting wax ink). To prepare the DCP, the circles were
filled with 10 μL of 0.10 mM dye solutions and dried for 1 h
at room temperature. The colors of the DCP were recorded with a smartphone.
The spots on DCP with different dyes were assigned as shown in
3
Protein Extraction and Immunoblotting
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Protein sample preparation and immunoblotting were performed as described previously5 (link)17 (link). In brief, tissue samples (i.e., embryos/larvae or eye tissues) were collected in chilled PBS and dissolved in lysis buffer [25 mM Tris, 150 mM NaCl, 1 mM EDTA·2 Na, 1% Igepal CA-630, 1% proteinase inhibitor cocktail (P-8340, Sigma, MO 63103, U.S.A), 1% sodium deoxycholate, and 0.1% SDS, pH 7.5] at a concentration of one tissue sample/7 µl lysis buffer. The supernatant was mixed with an equal volume of 2× sample buffer [100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.01% (w/v) bromophenol blue (Wako, Osaka, Japan), and 10% β-mercaptoethanol (Sigma)], boiled for 5 min, and then stored at −20°C until use. Proteins were separated on a 10% gel (456–1033; Mini-Protean TGX Gels; Bio-Rad, CA 94547, U.S.A) by SDS-PAGE and transferred onto a PVDF membrane (Immun-Blot PVDF membrane for protein blotting, Bio-Rad). The blots were conventionally labelled with the primary antibody and visualised with the biotinylated secondary antibody and an ABC/DAB system (Vectastain ABC Elite kit, DAB substrate kit, Vector Laboratories).
4
Inactivation of F. tularensis SCHU P9
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Five microliters of F. tularensis SCHU P9 was mixed with 100 μL of deionized water, 10%–90% ethanol (Sigma-Aldrich), 100% methanol (Nacalai Tesque, Inc., Kyoto, Japan), 100% acetone (Sigma-Aldrich), a mixture of 50% methanol and 50% acetone, 10% formaldehyde neutral buffer solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 4% PFA (Wako), 100% acetonitrile (Sigma-Aldrich) and final concentration of 0.001%–1% sodium hypochlorite (Purelux; Oyalox Co., Ltd., Tokyo, Japan), then incubated for 10 min at room temperature (23°C).
Five microliters of F. tularensis SCHU P9 was mixed with 100 μL of 1% Triton X-100, 1% NP-40 and 1% LDS (Nacalai Tesque) buffer supplemented with 1 × sodium dodecyl sulphate buffer, 10% glycerol (Wako) and 0.005% bromophenol blue (63 mM Tris-HCl, pH 6.8; Wako). These mixtures of detergents and bacteria were incubated at 4°C for 10 min, 1 h, and 24 h. All samples were centrifuged at 12,000 × g for 2 min at 4°C. Afterward, the supernatant was discarded to remove the effects of solvents. After the bacterial pellets were resuspended in 100 μL of CDM, the viable bacteria were counted. These experiments were conducted using four replicates.
Five microliters of F. tularensis SCHU P9 was mixed with 100 μL of 1% Triton X-100, 1% NP-40 and 1% LDS (Nacalai Tesque) buffer supplemented with 1 × sodium dodecyl sulphate buffer, 10% glycerol (Wako) and 0.005% bromophenol blue (63 mM Tris-HCl, pH 6.8; Wako). These mixtures of detergents and bacteria were incubated at 4°C for 10 min, 1 h, and 24 h. All samples were centrifuged at 12,000 × g for 2 min at 4°C. Afterward, the supernatant was discarded to remove the effects of solvents. After the bacterial pellets were resuspended in 100 μL of CDM, the viable bacteria were counted. These experiments were conducted using four replicates.
5
Tick Sample Protein Separation
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The prepared tick sample was mixed with sample buffer, i.e., 125 mM Tris-HCl, pH 6.8, 20% v/v glycerol, 4% w/v SDS, 10% v/v 2-mercaptoethanol, and a few crystals of bromophenol blue (Wako, Wako, Japan), and then boiled for 5 min. After sample loading (15 μg/lane), protein was separated on 12.5% polyacrylamide gel with discontinuous buffer system of pH 6.8 and 8.8 by constant current of 24 mA.
6
Electrophoretic Analysis of Microvesicles
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Prior to electrophoretic analysis, MVs and the culture supernatants were diluted with an equal volume of native-PAGE or SDS-PAGE buffer (0.06 M of Tris-HCl (Amersham Pharmacia Biotech, Buckinghamshire, UK), pH 6.8, 20% glycerol (Wako Pure Chemical Industries Ltd., Osaka, Japan), 0% or 1% (wt/vol) SDS (Wako), 0% or 1% 2-mercaptoethanol (2-ME, Merck kGaA), and 0.0012% bromophenol blue (Wako)). The SDS-PAGE samples were heated at 100 °C for 5 min just prior to loading on the gel. The native-PAGE or SDS-PAGE samples were then ran on a 12.5% polyacrylamide gel (e-PAGEL, ATTO Corp., Tokyo, Japan) in 0.025 M of Tris-HCl, 192 mM of glycine (Wako) and 0% or 0.1% (wt/vol) SDS. Electrophoretic separation of the proteins was carried out for 70 min at 25 mA. Gels were stained with Coomassie Brilliant Blue (CBB) and ethidium bromide to observe the proteins and DNA, respectively.
7
Polypyrrole Influence on S. mutans Proteins
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Before SDS-PAGE, culture supernatants (experiment 1 and 2) from S. mutans UA159 were cultivated with and without 2.5 mg/ml polypyrrole for 1 h at 37°C. Prior to electrophoretic analysis, the control, which contained PBS mixed with 2.5 mg/ml polypyrrole, and the culture supernatants with and without 2.5 mg/ml polypyrrole were diluted with an equal volume of SDS-PAGE sample buffer {0.06 M Tris-HCl (Amersham Pharmacia Biotech, Buckinghamshire, UK), pH 6.8; 20% glycerol (Wako Pure Chemical Industries Ltd, Osaka, Japan); 1% (vt/vol) SDS (Wako); 1% 2-mercaptoethanol (2-ME, Sigma-Aldrich); and 0.0012% bromophenol blue (Wako)}. The SDS-PAGE samples were heated at 100°C for 3 min prior to loading on the gel. The samples were then run on a 12.5% polyacrylamide gel (e-PAGEL, ATTO Corp., Tokyo, Japan) in the presence of 0.025 M Tris-HCl, 192 mM glycine (Wako) and 0.1% (wt/vol) SDS. Electrophoretic separation of the proteins was carried out for 70 min at 40 mA. Gels were stained with Coomassie blue to observe the proteins.
8
SDS-PAGE and Western Blot Analysis of SEA
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SDS-PAGE and Western blot analysis were performed as previously reported with some modifications [23 (link)]. Briefly, equal amounts of the supernatants and the sample buffer (250 mM Tris, pH 7, 4% SDS, 20% glycerol, 10% β-mercaptoethanol and 0.05% bromophenol blue; Wako Pure Chemical Industries, Ltd., Osaka, Japan) were treated at 110°C for 5 min. Then, 15 μL aliquots were evaluated with SDS–PAGE on a 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel. After electrophoresis, the protein bands were transferred to a polyvinylidene difluoride membrane. The membranes were probed with 1:20,000 (v/v) of rabbit anti-SEA IgG (Sigma, St. Louis, MO, USA, Lot Number 103K4831) and then reacted with 1:500 (v/v) of goat anti-rabbit peroxidase (KPL). SEA combined with the membrane was evaluated using a Protein Detector Western Blot Kit BCIP/NBT System (KPL, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The results were quantified with Image J software (National Institutes of Health, Bethesda, MD, USA).
9
Denaturing Polyacrylamide Gel Electrophoresis of Hemoglobin
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Tris(hydroxymethyl)aminomethane (Tris), sucrose, SDS, 2-mercaptoethanol, and bromophenol blue used to prepare Hb solutions for electrophoresis were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). A modified Hb solution (0.050 mM, 0.32 g/dL) was mixed with a half volume of a denaturing buffer 34 (0.19 M Tris-HCl pH 6.8, 6.0% (w/v) SDS, 15% (v/v) 2-mercaptoethanol, 30% (w/v) sucrose, and 0.006% (w/v) bromophenol blue)
and was incubated at 80 °C for 15 min. Electrophoresis was conducted on a 13% polyacrylamide gel using a mini-slab electrophoresis system (NA-1010; Nihon Eido Corp., Tokyo, Japan). A kit (Amersham Low Mw Calibration Kit for SDS Electrophoresis; GE Healthcare UK Limited, Buckinghamshire, UK) was used for Mw markers. The gel was stained with Coomassie brilliant blue (Quick CBB; Wako Pure Chemical Industries Ltd.). The stained gel image was obtained by transmission scanning (GT-X970; Seiko Epson Corp., Nagano, Japan).
and was incubated at 80 °C for 15 min. Electrophoresis was conducted on a 13% polyacrylamide gel using a mini-slab electrophoresis system (NA-1010; Nihon Eido Corp., Tokyo, Japan). A kit (Amersham Low Mw Calibration Kit for SDS Electrophoresis; GE Healthcare UK Limited, Buckinghamshire, UK) was used for Mw markers. The gel was stained with Coomassie brilliant blue (Quick CBB; Wako Pure Chemical Industries Ltd.). The stained gel image was obtained by transmission scanning (GT-X970; Seiko Epson Corp., Nagano, Japan).
10
Purification and Characterization of Hemoglobin Derivatives
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Human Hb used for this work was purified from RBCs provided by the Japanese Red Cross. As reported elsewhere in the literature, 29 a highly purified carbonyl Hb (native Hb) solution was obtained through pasteurization and nanofiltration. DBBF crosslinked Hb (XLHb) was also prepared as reported in the literature. 26, 30 The Hb derivative concentrations were determined using Hemoglobin B-test Wako (Fujifilm Wako Pure Chemical Corp.). Sodium chloride, potassium chloride, disodium hydrogen phosphate (Na 2HPO4), potassium dihydrogen phosphate (KH2PO4), polyethylene glycol 20,000 (20 kDa PEG), ammonium sulfate, Tris(hydroxymethyl) aminomethane (Tris), sucrose, sodium dodecyl sulfate (SDS), 2-mercaptoethanol, bromophenol blue, and Coomassie brilliant blue (Quick CBB) were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan). Saline (Otsuka normal saline) was purchased from Otsuka Pharmaceutical Co. Ltd. (Tokyo, Japan). Albumin (25% human serum albumin) was purchased from Japan Blood Products Organization (Tokyo, Japan).
Bi-functional maleimide PEG, mal2-PEG (Figure 1; SUNBRIGHT DE-100MA, Lot M153536, Mn 10456, Mw/Mn = 1.02, substitution 90.6%) was purchased from NOF Corp. (Tokyo, Japan).
Bis-(3,5-dibromosalicyl) fumarate (Figure 1; DBBF) was purchased from Abcam plc.
(Cambridge, U.K.).
Bi-functional maleimide PEG, mal2-PEG (Figure 1; SUNBRIGHT DE-100MA, Lot M153536, Mn 10456, Mw/Mn = 1.02, substitution 90.6%) was purchased from NOF Corp. (Tokyo, Japan).
Bis-(3,5-dibromosalicyl) fumarate (Figure 1; DBBF) was purchased from Abcam plc.
(Cambridge, U.K.).
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