All experiments were performed on a Superdex S75 10/300 GL column (GE Healthcare) with a sample injection volume of 250 μL and a flow rate of 0.5 mL/min at 4°C. The volume of the sample loop was 100 μL. Molecular weight markers used to calibrate the column were: albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) from the Low Molecular Weight Gel-filtration Calibration kit (GE Healthcare). Absolute molecular weight calculations were obtained by static light scattering in line with size exclusion chromatography using the Wyatt Optilab T-rEX refractometer and miniDAWN Treos multiangle light scattering system at 4°C. Protein concentrations were monitored by the refractometer and light scattering unit directly after the gel filtration column. Absolute molecular weights were determined using ASTRA version 6.0 (Wyatt Technologies).
Low molecular weight gel filtration calibration kit
Manufactured by GE Healthcare
Sourced in United States
The Low Molecular Weight Gel Filtration Calibration Kit is a laboratory equipment product designed for the calibration and assessment of gel filtration columns. It provides a set of standard proteins with known molecular weights, enabling the user to determine the separation range and performance of their gel filtration system.
Automatically generated - may contain errors
Lab products found in correlation
Astra version 6, by Wyatt Technology (1 mentions)
Optilab t rex refractometer, by Wyatt Technology (1 mentions)
Superdex s75 10 300 gl column, by GE Healthcare (1 mentions)
Superose 6 increase 10 300 column, by GE Healthcare (1 mentions)
Astra 6, by Wyatt Technology (1 mentions)
Minidawn treos, by Wyatt Technology (1 mentions)
5 protocols using low molecular weight gel filtration calibration kit
1
Protein Molecular Weight Analysis
2
Oligomeric state analysis of proteins
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Oligomeric state of purified proteins was analysed using the high resolution Superdex 200 Increase 10/300 GL column. 0.5 ml of concentrated samples (10, 20 and 30 μM) were loaded on the column which was pre-equilibrated with the running buffer (20 mM HEPES pH 7.5 and 250 mM NaCl). A constant flow rate of 0.5 ml/min was used and UV absorbance was monitored simultaneously at 280 nm, 254 nm and 220 nm at ~ 15°C. Standard calibration curve for the column was prepared using low molecular weight gel filtration calibration kit (GE Healthcare). To study protein-protein complexes, purified proteins were mixed (in varying molar ratios) and incubated at 4°C for 1 h before conducting SEC and analytical ultracentrifugation (AUC) experiments. The elution fractions were analysed using SDS-PAGE.
3
Analytical Calibration Methods for Trace Elements
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The external calibration method was used for procedures 1 and 2. Calibration curves were constructed based on five points over the concentration ranges of: 0.2 µg L−1 to 5.0 µg L−1 for AsIII and AsV, 0.1 µg L−1 to 5.0 µg L−1 for SbV, 0.5 µg L−1 to 5.0 µg L−1 for SbIII and CrVI for procedure 1, and 0.5 µg L−1 to 10.0 µg L−1 for all species analyzed in procedure 2. The average signal intensity (peak area value) of three replicates for each calibration standard was taken to construct the calibration curves [36 (link),37 (link)].
In procedure 3, retention times were calibrated using a Low Molecular Weight Gel Filtration Calibration Kit (GE Healthcare, Marlborough, MA, USA) consisting of five proteins with MW in the range 6500 to 75,000 Da. Retention times were determined using a UV/VIS detector in place of ICP-DRC-MS [35 (link),41 (link)].
In procedure 3, retention times were calibrated using a Low Molecular Weight Gel Filtration Calibration Kit (GE Healthcare, Marlborough, MA, USA) consisting of five proteins with MW in the range 6500 to 75,000 Da. Retention times were determined using a UV/VIS detector in place of ICP-DRC-MS [35 (link),41 (link)].
4
Oligomeric Status Determination of Fusion Proteins
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To determine the oligomeric status of the purified fusion proteins, size exclusion chromatography (SEC) was performed in non-denaturing conditions. 500 µL of the proteins ranging from 2 mg/mL – 4 mg/mL concentration were loaded on an analytical Superose 6 Increase 10/300 column (GE Healthcare) and eluted in 1x PBS (pH 7.4) at 0.4 mL/min flow rate on an Äkta pure chromatography system. A low molecular weight gel filtration calibration kit (product code-28403841) (GE Healthcare) was used for calibrating the column. For SEC-MALS (SEC-Multiangle light scattering), proteins were separated on a Superose 6 Increase 10/300 column (GE Healthcare) in 1x PBS (pH 7.4) at a flow rate of 0.4 mL/min. SEC resolved peaks were subjected to an in-line MALS detector (mini-DAWN TREOS, Wyatt Technology corp.) and a refractive index monitor (WATERS corp.) for molecular weight estimation. The data was analyzed through ASTRA 6.0 software (Wyatt Technology), as described previously (31 (link)).
5
Preparation of Trace Metal Standards
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All working solutions were prepared from high purity standards. AsIII, AsV, and CrVI working solutions were prepared from liquid standards by dilution to the desired concentration with ultrapure water. AsB, DMA, MMA, SbIII, and SbV working solutions were prepared from solid high purity standards by dissolving in ultrapure water. SEC column retention time calibration solutions were prepared from a Low Molecular Weight Gel Filtration Calibration Kit (GE Healthcare, Marlborough, MA, USA) by dissolving in phosphate buffer as suggested by the manufacturer.
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