The homogeneity and molecular weight distribution of the purified polysaccharideenriched extracts were estimated by high performance size exclusion chromatography (HPSEC) using two different columns (300 X 7.8 mm i.d., Tosoh Bioscience LLC, King of Prussia, PA) in sequence TSKgel GMPWXL (dextran MW<50000 kDa) and TSKgel G3000PWXL (dextran MW<60 kDa) as described previously. 22 The system was calibrated with standard dextrans of 252, 110, 70, 40, and 6 kDa, and glucose, using a regression curve.
Tskgel g3000pwxl
Manufactured by Tosoh
Sourced in Japan, Germany, United States
The TSKgel G3000PWXL is a size exclusion chromatography column designed for the analysis of macromolecules. It features a silica-based packing material with a pore size suitable for the separation of proteins, polymers, and other high-molecular-weight compounds.
Automatically generated - may contain errors
Lab products found in correlation
Tskgel g2500pwxl, by Tosoh (5 mentions)
Tskgel gmpwxl, by Tosoh (3 mentions)
G2500 pwxl, by Tosoh (3 mentions)
G4000pw xl columns, by Tosoh (2 mentions)
Refractive index detector, by Agilent Technologies (2 mentions)
Bicinchoninic acid protein assay kit bca kit, by Merck Group (1 mentions)
Shodex standard p 82, by Resonac (1 mentions)
Lc 10a, by Shimadzu (1 mentions)
Refractive index detector, by Shimadzu (1 mentions)
λ dna molecular weight marker 3 0.12 21 2 kbp, by Roche (1 mentions)
Tsk gel pwxl guard column, by Tosoh (1 mentions)
Redifrac fraction collector, by GE Healthcare (1 mentions)
Gpc system, by Jasco (1 mentions)
Rotary evaporator, by Heidolph (1 mentions)
Sephadex g 10 column, by Cytiva (1 mentions)
Vp ods column, by Shimadzu (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Poly ethylene oxide, by Tosoh (1 mentions)
1260 infinity, by Agilent Technologies (1 mentions)
Tsk gel g5000pwxl, by Tosoh (1 mentions)
25 protocols using tskgel g3000pwxl
1
Polysaccharide Characterization by HPSEC
2
Aqueous RAFT Synthesis of Sulfobetaine Polymers
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Sulfobetaine homopolymers were synthesized by aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. Typically, the MASB monomer was dissolved in pure water, followed by the chain transfer agent (CTA), 4-(2carboxyethylsulfanylthiocarbonyl)sulfanyl-4-cyanopentanoic acid. The initiator, 2,2 0 -azobis[2-(2-imidazolin-2-yl)propane] was dissolved in methanol and mixed with the monomer/CTA solution. The molar ratio of monomer to CTA and initiator was [monomer] :
[CTA] : [initiator] = 100 : 1 : 0.3, and the final concentration of the monomer was set to 0.1 M. The solvent was a mixture of pure water and methanol at a volume ratio of 2 : 1. Nitrogen gas was bubbled for 30 min to remove dissolved oxygen, and the polymerization was carried out in an oil bath at 60 1C for 18 h. The polymer was purified by dialysis in pure water (MWCO = 3500) for 7 days to remove any unreacted monomer. The polymer powder was obtained by freeze-drying the purified solution. The molecular weight of the polymers was determined via gel permeation chromatography using a JASCO system (Tokyo, Japan) with TSKgel G3000PW XL and G4000PW XL columns (Tosoh Co. Tokyo, Japan). The eluent was aqueous NaNO 3 (400 mM) calibrated with PEG standards.
[CTA] : [initiator] = 100 : 1 : 0.3, and the final concentration of the monomer was set to 0.1 M. The solvent was a mixture of pure water and methanol at a volume ratio of 2 : 1. Nitrogen gas was bubbled for 30 min to remove dissolved oxygen, and the polymerization was carried out in an oil bath at 60 1C for 18 h. The polymer was purified by dialysis in pure water (MWCO = 3500) for 7 days to remove any unreacted monomer. The polymer powder was obtained by freeze-drying the purified solution. The molecular weight of the polymers was determined via gel permeation chromatography using a JASCO system (Tokyo, Japan) with TSKgel G3000PW XL and G4000PW XL columns (Tosoh Co. Tokyo, Japan). The eluent was aqueous NaNO 3 (400 mM) calibrated with PEG standards.
3
Characterization of Polysaccharide Structure
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Total uronic acid content was determined by a colorimetrical method [17 (link)] using glucuronic acid as a standard. Protein content was measured by a Bicinchoninic Acid Protein Assay Kit (BCA kit) (Sigma-Aldrich, 3050 Spruce Street, St Louis, MO 63103, USA) [18 (link)] using bovine serum albumin as a standard. Sulfate content was assayed using an ion chromatography method [19 (link)]. Molecular weight (Mw) was determined by a high-performance liquid chromatography, coupled with a refractive index detector (Agilent Technologies, Wilmington, DE, USA), with a column of TSKgel G3000PWXL (TOSOH, Tokyo, Japan). Aqueous Na2SO4 solution (0.1 mol/L) was used as the mobile phase and the flow rate was 0.5 mL/min. The temperature of the column was maintained at 35 °C. Dextrans were used as standards to calibrate the column [20 (link)].
4
Molar Mass Distribution Analysis of Dialyzed Liquid Phases
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The dialyzed liquid phases after water extraction process (in the absence of carrageenans) obtained at different temperatures were evaluated to determine the profile of molar mass distribution by High Performance Size Exclusion Chromatography (HPSEC). A High-Performance Liquid Chromatograph (HPLC) from Agilent (Germany) was required to analyze this feature. The equipment was provided by two columns from Tosoh Bioscience (Germany), both were placed in series (300 mm × 7.8 mm TSKGel G3000PWXL and 300 mm × 7.8 mm TSKGel G2500PWXL) and a guard-column was also positioned in front (PWX-guard column, 40 mm × 6 mm) fitted by a refractive index (RI) detector. Columns were working at 70 °C and the flow of the mobile phase (Milli-Q water) was 0.4 mL/min. In order to establish a pattern, dextrans (DX) at a molecular weight of between 1000 and 80,000 g/mol from Fluka (USA) were used.
5
Analyzing Molecular Weight Distribution
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The homogeneity and molecular weight distribution of the purified extracts were estimated by high performance size-exclusion chromatography (HPSEC) using two different columns (300 × 7.8 mm i.d., Tosoh Bioscience LLC, King of Prussia, PA, USA) in sequence TSK gel GMPWXL (dextran Mw < 50,000 kDa) and TSKgel G3000PWXL (dextran Mw < 60 kDa), as described previously [25 (link)]. The system was calibrated with standard dextrans of 500, 110, 70, 40, and 6 kDa.
6
Molecular Weight Profiling by HPSEC
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The molecular weight profiles of the liquid samples were assessed by high-performance size exclusion chromatography (HPSEC). The columns used were a pre-guard column PWX-guard (40 × 6 mm2) and the columns TSKGel G3000PWXL and G2500PWXL (300 × 7.8 mm2), both from Tosoh Bioscience (Stuttgart, Germany). A refractive index (RI) detector was used to obtain the chromatograms, with the operation conditions: 70 °C, Milli-Q water as the mobile phase, and 0.4 mL/min the flow rate. Dextrans from 1 kDa to 80 kDa were used as patterns.
7
Analysis of β-1,3:1,4-Glucan Molecular Weight
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Digestion of β-1,3:1,4-glucan with enzyme was performed using a reaction mixture (total volume, 1 mL) consisting of the enzyme, 0.3% (w/v) β-1,3:1,4-glucan, and 50 mM 3-morpholinopropanesulfonic acid-NaOH buffer (pH 6.5). The apparent molecular weight (Mr) of the β-glucan was estimated by high-performance liquid chromatography (HPLC) with a Shimadzu LC-10 A (Shimadzu, Kyoto, Japan) fitted with a refractive index detector (Shimadzu) and tandem columns (each 7.8 × 300 mm) of TSKgel G3000PWXL and G2500PWXL (Tosoh, Tokyo, Japan), equilibrated and eluted with 0.2 M potassium phosphate buffer (pH 6.9) at a flow rate of 0.8 mL/min and at 40 °C. The void volume (V0) and inner volume (Vi) were determined with pullulan markers (Shodex Standard P-82; Showa Denko, Tokyo, Japan) and Glc.
8
Gel Filtration Analysis of Protein Conjugates
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Conjugate, free protein and free GACox samples were eluted on a TSK gel G3000 PWXL (30 cm × 7.8 mm) column (particle size 7 µm) with TSK gel PWXL guard column (4.0 cm × 6.0 mm; particle size 12 µm) (TosohBioscience, King of Prussia, PA, USA). The mobile phase was 0.1 M NaCl, 0.1 M NaH2PO4, 5% CH3CN, pH 7.2 at the flow rate of 0.5 mL/min (isocratic method for 35 min). Sample volume of injection was 80 µL. Void and bed volume calibration was performed with λ-DNA (λ-DNA Molecular Weight Marker III 0.12–21.2 Kbp, Roche, Risch-Rotkreuz, Switzerland) and sodium azide (NaN3, Merck, Darmstadt, Germany), respectively. GACox peaks were detected by refractive index (RI). Protein and conjugate peaks were also detected using tryptophan fluorescence (emission spectrum at 336 nm, with excitation wavelength at 280 nm). For the Kd determination the following equation was used:
where: Te = elution time of the analyte, T0 = elution time of the bigger fragment of λ-DNA and Tt = elution time of NaN3.
where: Te = elution time of the analyte, T0 = elution time of the bigger fragment of λ-DNA and Tt = elution time of NaN3.
9
Molecular Weight Analysis of Fraction B
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Analliquot of Fraction B was freeze-dried and redissolvedin 0.05 Mtris-hydrochloric acid. The method used for HPSEC analysis was that described by Jiménez et al. (Jiménez, Rodríguez, Fernández-Caro, Guillén, Fernández-Bolaños, & Heredia, 2001b ) with slight modifications. The MW was measured in Jasco equipment (LC-Net II ADC, Kyoto, Japan) with a refractive index detector (Jasco RI-1530) and injection valve (Rheodyne, loop 20 µL, Cotati, CA). Two different columns in sequence were used: TSKgelGMPWxl and TSKgel G3000PWxl (300 x 7.8 mmi.d., Tosoh Bioscience LLC, King of Prussia, PA) after calibration with 500, 110, 40, 6 kDa and maltose (Fluka, Buchs, Switzerland) . Blue dextran was used to test the void volume (V0) of the column. The elution buffer was 0.05 Mtris-hydrochloric acid at a flow rate 0.4 mL/min.Fractions of 250 L were collected using a Redifrac® fraction collector (Pharmacia Biotech, Uppsala, Sweden). Fractions were assayed for neutral sugars by the anthronemethod.
10
Molecular Weight Analysis of Purified Proteins
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The purified proteins and protein molecular weight standards (GE Healthcare, Little Chalfont, UK) were analysed using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA) with an analytical TSK Gel G3000PWXL (TOSOH, Tokyo, Japan) to analyse the molecular weight. In the system, the elution buffer was PBS + 0.01% SDS or 0.4 M NaCl with a flow rate maintained at 0.5 mL/min, and the absorbance at 280 nm was monitored for the detection of protein in the eluent.
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