The oligomeric state of the antidin sbAvd-7 was analyzed with size exclusion chromatography (SEC) using a liquid chromatography instrument (CBM-20A, Shimadzu Corporation) equipped with an autosampler (SIL-20A), UV-Vis (SDP-20A), and a fluorescence detector (RF-20Axs). The instrument was integrated with a static light scattering instrument (SLS, Zetasizer μV light scattering detector (Malvern Instruments Ltd.)) to determine molecular weight of the eluted proteins. The instrument was controlled using Lab Solutions Version 5.51 (Shimadzu Corporation) and OmniSEC 4.7 (Malvern Instruments Ltd.). Samples (~50 μg in 10–100 μl) were injected onto a Superdex200 Increase 5/150GL column (GE Healthcare) and equilibrated with the buffer the protein was dialyzed against (50 mM sodium phosphate, 650 mM NaCl, pH 7) with a flow rate of 0.1 ml/min at 20°C. Molecular weight determination was done by calculating a standard curve based on the elution volume of the molecular weight markers (CA, carbonic anhydrase 29 kDa; BSA, Bovine Serum Albumin 66 kDa; ADH, Alcohol Dehydrogenase 150 kDa; BA, ß-Amylase 200 kDa, Sigma-Aldrich), and alternatively, using the light-scattering intensity-based determination protocol involving BSA (monomeric peak) in SLS detector calibration using a Malvern microV detector and the OmniSEC software (Malvern Instruments Ltd.) (Fig 6 ).
Superdex 200 increase 5 150 gl column
Manufactured by GE Healthcare
Sourced in United States
The Superdex 200 Increase 5/150 GL column is a size exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. The column features a pre-packed Superdex 200 resin and is optimized for use on ÄKTA laboratory scale chromatography systems.
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Lab products found in correlation
Astra 6, by Wyatt Technology (3 mentions)
Labsolutions version 5, by Shimadzu (1 mentions)
Sil 20a, by Shimadzu (1 mentions)
Omnisec software, by Malvern Panalytical (1 mentions)
Omnisec 4, by Malvern Panalytical (1 mentions)
Cbm 20a, by Shimadzu (1 mentions)
Mwgf1000, by Merck Group (1 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
Agilent 1260 infinity 2 hplc system, by Agilent Technologies (1 mentions)
Refractive interferometric detection, by Wyatt Technology (1 mentions)
Optilab t rex system, by Wyatt Technology (1 mentions)
Astra software, by Wyatt Technology (1 mentions)
Minidawn treos detector, by Wyatt Technology (1 mentions)
Prominence lc 20ad, by Shimadzu (1 mentions)
Akta protein purification system, by GE Healthcare (1 mentions)
Hygromycin b, by Solarbio (1 mentions)
25 protocols using superdex 200 increase 5 150 gl column
1
Oligomeric State Analysis of Antidin sbAvd-7
2
Protein Molecular Weight Determination
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Analytical size-exclusion chromatography was performed on a Superdex 200 Increase 5/150 GL column (GE Healthcare) pre-equilibrated with storage buffer. Blue dextran (2000 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa) and ribonuclease A (13.7 kDa) were used as standards for column calibration. The Kav versus log molecular weight was calculated using the equation Kav = (Ve − V0)/(Vt − V0), where Ve is the elution volume of the protein, V0 is the void volume of the column and Vt is the column bed volume.
3
SAXS Analysis of SFTSV L Protein
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Small angle X-ray scattering (SAXS) of SFTSV L protein was performed with an in-line size exclusion chromatography on a Superdex 200 Increase 5/150 GL column (GE Healthcare) with a buffer containing 50 mM HEPES(NaOH) pH 7, 500 mM NaCl, 5% (w/v) glycerol and 2 mM dithiothreitol. Data was collected at the SAXS beamline P12 of PETRA III storage ring of the DESY, Hamburg, Germany (38 (link)) using a PILATUS 2M pixel detector at 3.0 m sample distance and 10 keV energy (λ = 1.24 Å), a momentum transfer range of 0.01 Å−1 < s < 0.45 Å−1 was covered (s = 4π sin θ/λ, where 2θ is the scattering angle). Data were analyzed using the ATSAS 2.8 package (39 (link)). The SEC-SAXS data were analyzed with CHROMIXS and the forward scattering I(0) and the radius of gyration Rg were extracted from the Guinier approximation calculated with the AutoRG function within PRIMUS (40 ). GNOM (41 ) provided the pair distribution function P(r) of the particle, the maximum size Dmax and the Porod volume. Ab initio reconstructions were generated with DAMMIF (42 (link)). Forty independent DAMMIF runs were compared and clustered into five main classes using DAMCLUST (43 (link),44 (link)). Models within each class were superimposed by SUPCOMB (45 ) and averaged using DAMAVER (46 ). The structures were visualized using UCSF Chimera (31 (link)).
4
Molecular Mass Analysis of YeaX and YeaW
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The native molecular mass of YeaX and YeaW was analyzed by size exclusion chromatography using a Superdex 200 increase 5/150 GL column (GE Healthcare). Protein standards (molecular weight marker kit MWGF1000, Sigma) in the presence of 10 mM Hepes-NaOH, pH 7.5, 150 mM NaCl at a flow rate of 0.45 ml min−1 were used for calibration. Samples of 20 µl (∼300 µM) were injected and elution was monitored at 280 and 445 nm.
5
SEC-MALS Analysis of RraA Proteins
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Size exclusion chromatography (Superdex 200 Increase 5/150 GL column, GE healthcare) equipped with multi-angle light scattering (MALS) instrument (WYATT, USA) was employed. The protein samples were applied to the Superdex 200 column in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5% glycerol, and 2 mM 2-mercaptoethanol. VvRraA1, VvRraA1-C9D, VvRraA2, VvRraA2-C9D, and E. coli RraA were diluted to 3 mg/ml in the corresponding buffer and then loaded onto the column. Data analyses were performed with ASTRA 6 software (WYATT, USA).
6
Coupling H-Ras to Maleimide Membranes
7
Molecular Weight Determination of Active Enzyme
8
Quantifying TLR8 Dimerization Kinetics
9
Optimizing Protein Complex Purification
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For measuring the effect of various components in the buffer on the purified protein complex, the following procedure was used. Solutions of purified protein complex at concentrations ranging from 2–4 μM (concentration was identical for testing a given set of conditions) in a volume of 100 μl of SEC buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 0.28% NG, 100 nM NT1), adjusted to the specified conditions or supplemented with the specified components as described in Fig 4 , were prepared. For testing the effect of GDP and GTPγS, the solutions were also supplemented with 0.1 mM MgCl2. The solutions were incubated at 4°C for a time-period ranging from overnight to seven days, depending on the conditions and as described in Fig 4 . Prior to loading on the column, the mixtures were centrifuged at 10,000× g for 10 min at 4°C. The analytical SEC runs were performed on a Superdex 200 Increase 5/150 GL column (GE Healthcare) pre-equilibrated in the same buffer. The elution profile was monitored by absorption at 280 nm.
10
Stoichiometry of DM64-Myotoxin II Interaction
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The stoichiometry of the interaction between DM64 and myotoxin II was analyzed by SEC, as described (Bastos et al., 2020 (link)), with modifications. A fixed amount of DM64 was titrated with increasing concentrations of myotoxin II (1:0.5; 1:1; 1:2; 1:4 mol/mol) in 0.1 M Tris-HCl pH 7.5, containing 0.5 M NaCl. The mixtures were incubated at 25°C for 15 min and then injected on a Superdex 200 Increase 5/150 GL column (GE Healthcare), previously equilibrated with the same buffer. The chromatogram peaks were integrated, and the areas corresponding to free myotoxin or to free DM64 co-eluted with DM64-myotoxin complex were plotted. The experiment was run in duplicate.
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