MALLS studies were performed in-line with SEC on mutant IgM protein samples to assess mono-dispersity and the molecular mass of the protein samples. The peaks corresponding to the homodimeric Fcμ2-4 mutants from SEC were run on the Superdex 200 Increase 5/150 column (GE Healthcare) using an in-line miniDAWN multi-angle light scattering detector and an Optilab DSP Interferometric Refractometer (Wyatt Technology). The data were analyzed using the ASTRA 4.9 software (Wyatt Technology).
Superdex 200 increase 5 150
Manufactured by GE Healthcare
Sourced in Sweden
Superdex 200 Increase 5/150 is a pre-packed size exclusion chromatography column for analysis and purification of proteins, peptides, and other biomolecules. The column has a matrix of cross-linked agarose beads and a bed volume of 3 mL. It is designed for use with fast protein liquid chromatography (FPLC) systems.
Automatically generated - may contain errors
Lab products found in correlation
Optilab dsp interferometric refractometer, by Wyatt Technology (1 mentions)
Astra 4, by Wyatt Technology (1 mentions)
Agilent 1260 infinity lc chromatographic system, by Agilent Technologies (1 mentions)
Astra 6, by Wyatt Technology (1 mentions)
Optilab t rex refractive index detector, by Wyatt Technology (1 mentions)
Dawn heleos 2 8 angle, by Wyatt Technology (1 mentions)
Labsolution version 5, by Shimadzu (1 mentions)
Omnisec 4, by Malvern Panalytical (1 mentions)
Cbm 20a, by Shimadzu (1 mentions)
Astra 8, by Wyatt Technology (1 mentions)
Superdex 200 increase 10 300, by GE Healthcare (1 mentions)
4 protocols using superdex 200 increase 5 150
1
Characterization of IgM Mutants by MALLS
2
Size-Exclusion Chromatography of Mycobacterial Proteins
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Samples of Mtb-SecBTA at 49.1 µM (1.0 mg ml−1), Mtb-SecBTA/HigA1 at 6.5 µM (0.75 mg ml−1), and Mtb-HigA1ΔC42 at 100 µM (1.4 mg ml−1) in 20 mM MES, 200 mM NaCl, pH 6.5 were analyzed on a Superdex 200 Increase 5/150 (GE Healthcare) with multi-angle light scattering. The column was equilibrated with the same buffer, previously sterilized on a 0.1-µm filter, on an Agilent 1260 Infinity LC chromatographic system (Agilent Technology). Data were collected on a DAWN HELEOS-II 8-angle and Optilab T-rEX refractive index detector (Wyatt Technology, Toulouse, France). We loaded 75–100 μl of each protein sample and separation was performed at a flow rate of 0.4 ml min−1 at 15 °C. Results were analyzed with ASTRA 6.0.2.9 software (Wyatt Technology Corp.).
3
Protein Characterization by Liquid Chromatography
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Proteins were analyzed using a liquid chromatography instrument (CBM-20A; Shimadzu Corporation, Kyoto, Japan) equipped with an autosampler (SIL-20A), ultraviolet-visible (UV-VIS) (SPD-20A), and a fluorescence detector (RF-20Axs). Molecular weight was determined using SEC with a Malvern Zetasizer µV instrument (Malvern Instruments Ltd.) measuring inline SLS and DLS. Data were processed using Lab Solution Version 5.51 (Shimadzu Corporation) and OmniSec 4.7 (Malvern Instruments Ltd.) software. Samples (50 µg) were injected into the column (Superdex 200 Increase 5/150; GE Healthcare, Uppsala, Sweden) using the autosampler. The column was equilibrated with 50 mM Na3PO4, 150 mM NaCl, pH 7.2 running buffer. The measurements were run with a flow rate of 0.1 mL/min at 20 °C. The monomeric peak of bovine serum albumin (BSA) was used for calibrating the system to calculate molecular weight from the measured SLS intensity.
4
Absolute Mass Determination of N-Complexes
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To determine the absolute masses of various N constructs and their complexes with 14-3-3, we coupled a SEC column to a ProStar 335 UV/Vis detector (Varian Inc., Melbourne, Australia) and a multi-angle laser light scattering detector miniDAWN (Wyatt Technologies). Either Superdex 200 Increase 10/300 (~24 ml, flow rate 0.8 ml/min) or Superdex 200 Increase 5/150 (~3 ml, flow rate 0.45 ml/min) columns (GE Healthcare) were used. The miniDAWN detector was calibrated relative to the scattering from toluene and, together with concentration estimates obtained from UV detector at 280 nm, was exploited for determining the Mw distribution of the eluted protein species. All processing was performed in ASTRA 8.0 software (Wyatt Technologies) taking dn/dc equal to 0.185 and using extinction coefficients listed in Supplementary table 5. Protein content in the eluted peaks was additionally analyzed by SDS-PAGE.
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