GTC (Fluka, Buchs, Switzerland) and crowding agents PEG-600, PEG-8000, and PEG-12000, Dextran-70, and Ficoll-70 (Sigma-Aldrich, St. Louis, MO, USA) were used without additional purification. The absorbance of protein samples did not exceed 0.15 in all our experiments unless otherwise indicated. Taking into account the extinction coefficient ɛ280 = 31,519 M−1·sm−1 [19 (link)], this means that protein concentration was not higher than 5 µM. Measurements were conducted in 100 mM Na-phosphate-buffered solution at pH 8.0 in the absence or presence of different concentrations of crowding agents.
Dextran 70
Manufactured by Merck Group
Sourced in United States
Dextran 70 is a type of polysaccharide polymer derived from the bacterium Leuconostoc mesenteroides. It is a biocompatible and water-soluble substance commonly used in laboratory applications. Dextran 70 has a molecular weight of approximately 70 kilodaltons and is primarily used as a volume expander and as a component in various cell culture media and buffer solutions.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll 70, by Merck Group (3 mentions)
Peg 8000, by Merck Group (2 mentions)
Dextran 40, by Merck Group (2 mentions)
Softmax pro v5, by Molecular Devices (2 mentions)
Peg12000, by Merck Group (1 mentions)
Peg 600, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fla 7000 image analyzer, by Fujifilm (1 mentions)
Ficoll 70, by GE Healthcare (1 mentions)
Multi gauge software, by Fujifilm (1 mentions)
Tris base, by Bio-Rad (1 mentions)
Ecolite scintillation cocktail, by MP Biomedicals (1 mentions)
Triton x 100, by Bio-Rad (1 mentions)
Mn chloride tetrahydrate mncl2 4h2o, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Radioactive 14c sucrose, by Moravek Biochemicals (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Trisodium citrate solution, by Merck Group (1 mentions)
17 protocols using dextran 70
1
Protein Concentration Measurement in Crowded Environments
2
Microdialysis Probe Preparation and Perfusion
3
Erythrocyte Aggregation and Deformability
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Fresh venous blood samples were collected from patients and control subjects into 10 ml vacutainer tubes containing K2-EDTA. Briefly, whole blood was centrifuged at 1400 x g for 5 min and plasma was separated. For aggregation measurements, erythrocytes were prepared either with autologous plasma or isotonic phosphate buffered saline (PBS, 290 mOsm/kg, pH 7.4) containing 3% Dextran 70 (MW 70 kDa; Sigma Chemical Co., St. Louis, MO, USA) at a hematocrit (Hct) of 45%. For the measurements of erythrocyte deformability, the Hct of whole blood was adjusted to the level of 45%, as well.
4
Evaluating Protein Aggregation Propensity
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The method for the evaluation of the aggregation propensity followed those used in previous comprehensive analysis (Niwa et al., 2009 (link)). The template DNA for expression by the cell-free translation system was amplified from an E. coli ORF library [ASKA library (Kitagawa et al., 2005 (link); Riley et al., 2006 (link))] by PCR, as described previously (Niwa et al., 2009 (link)). The transcription-translation-coupled expression was conducted by a reconstituted cell-free translation system [PURE system (Shimizu et al., 2001 (link), 2005 (link))] at 37°C for 1 h. For detection, L-[35S]-methionine was added to the PURE system. Ficoll 70 (GE Healthcare) or dextran 70 (Sigma–Aldrich) was also included at the concentration of 80 mg/ml in the reaction, to evaluate the effects of MCRs. After the expression, an aliquot was withdrawn as the total fraction, and the remainder was centrifuged at 20,000 × g for 30 min. The total and supernatant fractions were separated by SDS-PAGE, and the band intensities were quantified by autoradiography (FLA7000 image analyzer and Multi Gauge software, Fujifilm). The ratio of the supernatant to the total protein was defined as the solubility, as referred to as the index of aggregation propensity.
5
Assessing Protein Interactions in Neurodegeneration
6
Plasmonic Biosensor Platform for HMGB1 Detection
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All chemical reagents are analytical-graded reagents. Hydrogen tetrachloroaurate trihydrate (HAuCl4), Phosphate buffered saline (PBS buffer), trisodium citrate solution (C6H5Na3O7, ≥99%), (3-Mercaptopropyl)methyldimethoxysilane (MPDMS, >95%), cystamine dihydrochloride (cystamine), dextran 70 (MW ≈ 70,000), sodium periodate (NaIO4), 11-mercaptoundecanoic acid (MUA; ≥95%), 6-mercapto-1-hexanol (MCH; ≥97%), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), ethanolamine (EA), mouse IgG, streptavidin, monoclonal anti-HMGB1 antibody, and high mobility group box 1 (HMGB1) protein were from Sigma-Aldrich (St. Louis, MO, USA). Sulfuric acid (H2SO4; ≥98%), hydrogen peroxide (H2O2), ethanol, and acetate buffer were purchased from Fluka (Buchs, Switzerland). Ultrapure deionized water (18.2 MΩ·cm−1, Milli-Q pure water purification system, Millipore Ltd., Burlington, MA, USA) was used for preparing solutions. The optical fiber probe was multimode plastic-clad silica optical fiber (model F-MBC, Newport), with core and cladding diameters of 400 and 430 μm, respectively, bought from Instant NanoBiosensors Co., Ltd. (Taipei, Taiwan). Sensing chips (poly (methyl methacrylate) (PMMA) plates) were prepared using a CO2 laser engraving machine (New Taipei, Taiwan).
7
Reagents for RNA Structure Probing
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PEG4000 was from Alfa Aesar; PEG8000 was from Research Organics; PEG200, proline, TMAO, betaine, Dextran10, Dextran70, and Ficoll70 were from Sigma-Aldrich; methanol was from Mallinckrodt. MgCl2 was obtained from J.T. Baker; KCl was from EMD Chemicals; and sodium cacodylate was from Sigma-Aldrich. 1M7 was a gift from the Showalter laboratory. Polynucleotide kinase was from New England Biolabs. SuperScript III reverse transcriptase was from Invitrogen.
8
Purification and Characterization of iRFP713
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iRFP713 and its variants in the holo- and apoforms were expressed and purified as described previously (Stepanenko et al., 2014 (link)) (Supplemental Methods 1 ). SDS/PAGE in a 12% polyacrylamide gels was used to confirm that the purity of the target proteins was at least 95% (Laemmli, 1970 (link)). The concentrated protein was stored in 20 mM Tris/HCl buffer, 150 mM NaCl, pH 8.0. The measurements were carried out at a low protein concentration (absorbance was kept less than 0.1, which corresponds to a protein concentration of 0.14 mg/ml) in 20 mM Tris/HCl buffer, pH 8.0, 1 mM tris(2-carboxyethyl)phosphine (TCEP).
GdnHCl, GTC, TCEP, and crowding agents PEG-8000, Dextran-40 and Dextran-70 were purchased from Sigma (St. Louis, MO, USA). The concentration of GdnHCl and GTC in stock solutions was calculated by the refraction coefficient measured by the Abbe refractometer (LOMO, St. Petersburg, Russia).
GdnHCl, GTC, TCEP, and crowding agents PEG-8000, Dextran-40 and Dextran-70 were purchased from Sigma (St. Louis, MO, USA). The concentration of GdnHCl and GTC in stock solutions was calculated by the refraction coefficient measured by the Abbe refractometer (LOMO, St. Petersburg, Russia).
9
Protein Purification and Crowding Solution Preparation
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Lyophilized BSA (Sigma–Aldrich, catalog number: A6003) was dissolved in Reaction buffer (50 mM Tris-HCl, pH7.5, 150 mM GluK, 5 mM GluMg) at approximately 100 g/L. Then, the buffer was exchanged with Reaction buffer to wash out residual molecules using Amicon Ultra-0.5 centrifugal filter unit 50 kDa. After washing steps, the concentration of BSA was measured by Bradford Assay. Ficoll70 and Dextran70 (Sigma–Aldrich) were dissolved in Reaction buffer, and their concentration was calculated from the weight of crowding agents and total volume of the solution (typically, the final concentration of the solution was 300–500 g/L). Crowding solutions were stored at −20 °C until further use.
10
Macromolecular Interactions Characterization
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