The Mw of polysaccharides was determined using high-performance size exclusion chromatography, as described previously by Xia et al. [15 (link)]. The instrument was equipped with a multi-angle laser light scattering system (DAWN, HELEOS, Wyatt Technology Co., Santa Barbara, CA, USA), connected with a refractive index detector (Waters-2414, Milford, MA, USA) and the MALLS system (Waters 2695, Milford, MA, USA), equipped with columns of TSK-gel G-3000 PW XL (300 × 7.8 mm, Tokyo, Japan) and TSK-gel G5000PWXL columns (300 × 7.8 mm, Tokyo, Japan). Briefly, the EHP solution (1 mg/mL) was passed through a 0.22 μm filter, and 20 μL of the sample was injected into the system. The experimental conditions were as follows: the mobile phase consisted of a 0.9% NaCl solution at a flow rate of 0.5 mL/min and 35 °C.
G3000 pwxl
Manufactured by Waters Corporation
Sourced in Japan, United States
The G3000 PWXL is a laboratory instrument manufactured by Waters Corporation. Its core function is to perform size exclusion chromatography (SEC) analysis of macromolecules such as proteins, polymers, and other large molecules.
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4 protocols using g3000 pwxl
1
Polysaccharide Molecular Weight Determination
2
Characterization of High-G/M and Mid-G/M Alginates
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Sodium alginate with molecular weight (Mw) of 520 kDa and G/M ratio of 64/36 was designated High-G/M alginate, and sodium alginate with Mw of 532 kDa and G/M ratio of 34/66 was designated Mid-G/M alginate. Both were purchased from Qingdao Mingyue Seaweed Group Co., Ltd. (Qingdao, China). The detailed information of both purchased alginates was characterized by GPC and 1HNMR spectroscopy (Table 1 ).
The G/M ratio of the respective alginates was determined at 80°C by 1HNMR spectroscopy using an AVANCE NB-360 spectrometer (Bruker, Germany). The Mw and molecular weight distribution (Mw/Mn) of alginate samples were analyzed via gel permeation chromatography (GPC) using a system equipped with two TSK-gel size-exclusion columns (G4000PWXL and G3000PWXL), a refractive index detector (Waters, model 2414, Milford, MA), and an HPLC pump (Waters, model 515).
Collagen, acquired from rat tails, and was obtained by our laboratory. Anhydrous calcium chloride (CaCl2) was purchased from Sigma-Aldrich (Merck Life Science (Shanghai) Co., Ltd.). All other chemical reagents were analytical grade and used as received.
The G/M ratio of the respective alginates was determined at 80°C by 1HNMR spectroscopy using an AVANCE NB-360 spectrometer (Bruker, Germany). The Mw and molecular weight distribution (Mw/Mn) of alginate samples were analyzed via gel permeation chromatography (GPC) using a system equipped with two TSK-gel size-exclusion columns (G4000PWXL and G3000PWXL), a refractive index detector (Waters, model 2414, Milford, MA), and an HPLC pump (Waters, model 515).
Collagen, acquired from rat tails, and was obtained by our laboratory. Anhydrous calcium chloride (CaCl2) was purchased from Sigma-Aldrich (Merck Life Science (Shanghai) Co., Ltd.). All other chemical reagents were analytical grade and used as received.
3
Protein and Carbohydrate Quantification of Extracellular Polymeric Substances
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The total protein content of EPS, IPS, EPSp, and IPSp was determined by the Bradford method, using the bovine serum albumin (BSA, BioFroxx, Guangzhou, China) as the standard [29 (link)]. The total carbohydrate content of all samples was determined by the total carbohydrate assay kit (Cat#BC2710, Solarbio, Shanghai, China) according to the instructions.
Molecular weight (MW) of EPS, IPS, EPSp, and IPSp was measured by an HPGPC instrument equipped with a Waters 1525 HPLC system and two consecutive TSK-GEL columns (G5000 PWXL and G3000 PWXL, 7.8 × 300 mm) (Waters Co, Milford, MA, USA) as previously reported [30 (link)]. All samples were dissolved in Milli-Q water (5 mg/mL) and centrifuged at 6000 rpm (EPPENDORF, 5427 R, rotor FA-45-30-11) for 15 min. The supernatant was collected and filtered through a 0.45 μM membrane prior to injection. The calibration curve was obtained using dextran MW standards with the peak MW of 1, 5, 12, 25, 50, 80, 270, 410, and 670 kDa.
Molecular weight (MW) of EPS, IPS, EPSp, and IPSp was measured by an HPGPC instrument equipped with a Waters 1525 HPLC system and two consecutive TSK-GEL columns (G5000 PWXL and G3000 PWXL, 7.8 × 300 mm) (Waters Co, Milford, MA, USA) as previously reported [30 (link)]. All samples were dissolved in Milli-Q water (5 mg/mL) and centrifuged at 6000 rpm (EPPENDORF, 5427 R, rotor FA-45-30-11) for 15 min. The supernatant was collected and filtered through a 0.45 μM membrane prior to injection. The calibration curve was obtained using dextran MW standards with the peak MW of 1, 5, 12, 25, 50, 80, 270, 410, and 670 kDa.
4
Graphene Oxide Synthesis and Cell Culture
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Graphene oxide (GO) was purchased from ACS Material (USA) and used after purification by dialysis tube with nominal molecular weight cut off 10 6 Da. SpectraPor dialysis tubing was purchased from Spectrum Laboratories, Inc. (Italy). 1 H NMR spectra were recorded using a Bruker Avance II 300 spectrometer operating at 300.12 MHz.
Size exclusion chromatography (SEC) was carried out using Tosho Bioscience TSK-Gel G4000 PWXL and G3000 PWXL columns connected to a Water 2410 refractive index detector. The mobile phase was a 0.1 M Tris buffer pH 8.10± 0.05 with 0.2 M sodium chloride. The flow rate was 0.8 mL min -1 and sample concentration 3 mg mL -1 and PEG standards (100-0.400 kDa, Polymer Laboratories Inc., USA) were used to set up calibration curve (R 2 = 0.996).
Human fibroblasts (HDFa), breast cancer MCF-7 and MDA-MB-231 cells (purchased from Istituto Zooprofilattico Sperimentale della Lombardia e dell' Emilia Romagna, Italy) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v Fetal Bovine Serum (FBS), 2 mM Lglutamine, 100 units/mL penicillin G, 100 µg/mL streptomycin at 37°C and 5% CO2.
Size exclusion chromatography (SEC) was carried out using Tosho Bioscience TSK-Gel G4000 PWXL and G3000 PWXL columns connected to a Water 2410 refractive index detector. The mobile phase was a 0.1 M Tris buffer pH 8.10± 0.05 with 0.2 M sodium chloride. The flow rate was 0.8 mL min -1 and sample concentration 3 mg mL -1 and PEG standards (100-0.400 kDa, Polymer Laboratories Inc., USA) were used to set up calibration curve (R 2 = 0.996).
Human fibroblasts (HDFa), breast cancer MCF-7 and MDA-MB-231 cells (purchased from Istituto Zooprofilattico Sperimentale della Lombardia e dell' Emilia Romagna, Italy) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v Fetal Bovine Serum (FBS), 2 mM Lglutamine, 100 units/mL penicillin G, 100 µg/mL streptomycin at 37°C and 5% CO2.
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