We configured a Biorad Biological DuoFlow 10 system coupled with a miniDAWN-TREOS static 3-angle laser light-scattering detector and an Optilab-rEX refractive index detector (Wyatt Technology) for conducting the SEC-MALS experiments. 250 µl of purified protein sample (0.1–0.5 mg) was passed, at a constant flow rate of 0.5 ml/min, through an ENrich SEC 650 10 × 300 high-resolution column (Biorad) thoroughly pre-equilibrated with buffer B at room temperature. UV absorbance was measured with the detector at 280 nm. Light scattering and refractive index were monitored at a wavelength of 658 nm. BioLogic DuoFlow software version 5.3 (Biorad) was used to control the chromatography system, and Astra 5.3.4 software (Wyatt Technology) was used for data collection and analysis. The light-scattering (LS) detectors were normalized with monomeric BSA (Sigma). Baseline settings for all laser detectors as well as peak alignment and band-broadening correction between the UV, LS, and refractive index detectors were performed using Astra software algorithms. Data were processed to determine the weight-average molar mass and polydispersity of the protein sample using the Debye model as per the manufacturer’s instructions. A minimum of three repeat runs was conducted for each construct under identical experimental conditions.
Monomeric bsa
Manufactured by Merck Group
Monomeric BSA is a laboratory reagent that consists of purified bovine serum albumin (BSA) in its monomeric form. BSA is a widely used protein in various biochemical and cell culture applications due to its stability, solubility, and ability to maintain the activity of sensitive biomolecules. The monomeric form of BSA is commonly used as a standard, blocking agent, or additive in techniques such as Western blotting, ELISA, and cell culture media.
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Lab products found in correlation
Optilab t rex, by Wyatt Technology (9 mentions)
Dawn heleos 2, by Wyatt Technology (6 mentions)
Heleos 2, by Wyatt Technology (4 mentions)
Superdex 200 increase 10 300 column, by GE Healthcare (4 mentions)
Optilan t rex, by Wyatt Technology (3 mentions)
Astra 6, by Wyatt Technology (2 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (2 mentions)
Enrich sec 650 10 300 high resolution column, by Bio-Rad (1 mentions)
Optilab rex refractive index detector, by Wyatt Technology (1 mentions)
Astra 5, by Wyatt Technology (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300 size exclusion column, by GE Healthcare (1 mentions)
Dawn heleos 2 mals detector, by Wyatt Technology (1 mentions)
Optilab t rex differential refractometer, by Wyatt Technology (1 mentions)
Wtc 030s5 size exclusion column, by Wyatt Technology (1 mentions)
Superdex 200 increase 10 300, by GE Healthcare (1 mentions)
Kaleidagraph, by Synergy Software (1 mentions)
Astra software package, by Wyatt Technology (1 mentions)
Tskgel g3000swxl column, by Tosoh (1 mentions)
Optilab rex, by Wyatt Technology (1 mentions)
18 protocols using monomeric bsa
1
SEC-MALS Characterization of Protein Samples
2
SEC-MALS Analysis of Bacterial Proteins
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Purified BpFL (4 mg/ml), XccFL (4 mg/ml), BpCTR (6 mg/ml) and XccCTR (6 mg/ml) were subjected to SEC using a Superdex 200 10/300 gl (GE) column equilibrated in SEC-MALS buffer (20 mM HEPES pH7.5, 150 mM NaCl, 5 mM MgCl2 , 1 mM DTT). The gel filtration column was coupled to a static 18-angle light scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX) (Wyatt Technology). Data were collected continuously at a flow rate of 0.5 ml/min. Data analysis was performed using the program Astra VI. Monomeric BSA (6.0 mg/ml) (Sigma) was used for normalization of the light scattering detectors and data quality control.
3
Oligomeric State Analysis of TLK2 Proteins
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The oligomeric state of TLK2 proteins, including the heterodimers, was analyzed by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). Protein samples were prepared at 1 mg/ml concentration and dialyzed into gel filtration running buffer, 20 mM Tris-HCl, 150 mM NaCl, 0.5 mM TCEP, pH 8.0. The samples were loaded on a Superdex 200 Increase 10/300 size exclusion column (GE Healthcare). The column outlet was directly connected to a DAWN HELEOS II MALS detector (Wyatt Technology) followed by an Optilab T-rEX differential refractometer (Wyatt Technology). Data were collected and analyzed using ASTRA 6 software (Wyatt Technology). Samples were run in triplicates. The monomeric BSA was used as standard (Sigma-Aldrich).
4
Protein Molecular Weight Determination
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5 mg/ml (0.24 mM) of purified protein (wild-type OrnVc, OrnVc-D12A, or OrnVc-R130A) was injected onto a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated with gel filtration buffer (25 mM Tris-Cl, pH 7.5, 150 mM NaCl). Samples were run continuously at a flow rate of 0.75 ml/min through the gel filtration column coupled to a static 18-angle light scattering detector (DAWN HELIOS-II) and a refractive index detector (Optilab T-rEX), with data being collected every second. Data analysis was performed with ASTRA VI, yielding the molar mass and mass distribution (polydispersity) of the sample, using monomeric BSA (Sigma; 5 mg/ml) to normalize the light scattering detectors and as a control sample.
5
Characterizing Glutaminase C Oligomerization
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Example 7
Purified GAC and GAC mutants were subjected to multi-angle light scattering (MALS) as previously described by Moller et al., “Small Angle X-Ray Scattering Studies of Mitochondrial Glutaminase C Reveal Extended Flexible Regions, and Link Oligomeric State with Enzyme Activity,” PLoS One 8(9):e74783 (2013), which is hereby incorporated by reference in its entirety. Briefly, 50 μL samples of 5 mg/mL GAC were injected onto a WTC-030S5 size-exclusion column (Wyatt technology), coupled to a static 18-angle light scattering detector (DAWN HELEOS-II) and a refractive index detector (OptiLab TrEX, Wyatt Technology), at 23° C. The size-exclusion column was equilibrated with 20 mM Tris-HCl, pH 8.5, and 200 mM NaCl with or without 50 mM K2HPO4. The flow rate was kept at 1 mL/minutes. RMS radius and mass distribution (polydispersity) were analyzed using the ASTRA software, with monomeric BSA (Sigma) serving to normalize the light scattering signal.
6
SEC-MALS Analysis of Vps1 Oligomerization
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Purified S. cerevisiae Vps1 G436D (4.0 mg/ml) was subjected to SEC using a Superdex 200 10/300 GL equilibrated in SEC-MALS buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 5.0 mM MgCl2, 2.0 mM EGTA, and 2.0 mM β-mercaptoethanol). C. thermophilum Vps1 GG (2.5 mg/ml) and Vps1 GG K56A (4.0 mg/ml) were subjected to SEC using a Superdex 75 10/30 equilibrated in SEC-MALS buffer. Nucleotide-dependent oligomerization was assessed by incubating with various nucleotides at 2.0 mM for 1 h at 37°C followed by centrifugation at 13,000 g for 10 min before SEC-MALS analysis. The gel filtration column was coupled to a static 18-angle light-scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX; Wyatt Technology). Data were collected continuously at a flow rate of 0.5 ml/min. Data analysis was performed using the program Astra VI. Monomeric BSA (6.0 mg/ml; Sigma-Aldrich) was used for normalization of the light-scattering detectors and data quality control.
7
Size-Exclusion Chromatography of Pib2 FYVE
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After filtration through a 0.22 µm cellulose acetate membrane, the Pib2 FYVE constructs and mutants were subjected to size-exclusion chromatography using a Superdex 75 10/300 equilibrated in SEC-MALS buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl and 1.93 mM β-mercaptoethanol) at room temperature. 500 µl of each protein was loaded onto the column, at a concentration of 50 or 100 µM as indicated. The column was coupled to a static 18-angle light scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX) (Wyatt Technology). Data were collected continuously at a flow rate of 0.3 ml/min, with the flow cells in the scattering and refractive index detectors set to 25°C. Data analysis was performed using the program Astra VII. Monomeric BSA (2.0 mg/ml) (Sigma) was used for data quality control.
8
Size-Exclusion Chromatography and Light Scattering Analysis
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Purified, wild-type or mutant variant NrnCBh at 2 mg/ml (85 µM) was injected onto a Superdex 200 Increase 10/300 gel filtration column (GE Healthcare) equilibrated with gel filtration buffer (25 mM Tris-Cl, pH 7.5, 150 mM NaCl). Size-exclusion chromatography was coupled to an in-line, static 18-angle light scattering detector (DAWN HELEOS-II, Wyatt Technology) and a refractive index detector (Optilab T-rEX, Wyatt Technology). Data were collected every second. Data analysis was performed with Astra 6.1 (Wyatt Technology) yielding the molar mass and mass distribution (polydispersity) of the sample. Monomeric BSA (Sigma; 5 mg/ml) was used as a control sample and to normalize the light scattering detectors.
9
Oligomeric State Analysis of PLRV and TuYV N-Terminal Domains
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The oligomeric state of PLRV and TuYV NRTD was determined by size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). The NRTD proteins were loaded at 4 mg/mL onto a Superdex 200 10/300 Increase column (GE) in size exclusion buffer (20 mM HEPES pH 7.5, 150 mM KCl, and 1 mM DTT) at a flow rate of 0.5 ml/min. Eluent from the sizing column flowed directly to a static 18-angle light scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilan T-rEX) (Wyatt Technology) with data collected every second. Molar mass was determined using the ASTRA VI software. Monomeric BSA (Sigma) was used for normalization of light scattering.
10
Size-exclusion Chromatography of RexA
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Purified RexA constructs and homologs at 4 mg/ml were subjected to size-exclusion chromatography using a Superdex 200 10/300 GL Increase column (Cytiva) equilibrated in SEC buffer (20 mM HEPES pH 7.5, 150 mM KCl, and 1mM DTT). The column was coupled to a static 18-angle light scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX) (Wyatt Technology). Data were collected continuously at a flow rate of 0.6 ml/min. Data analysis was carried out using the program Astra VI and graphs generated using Kaleidagraph (Synergy Software). Monomeric BSA at 5 mg/ml (Sigma) was used for normalization of the light scattering detectors and data quality control.
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