The mass measurements were performed on a Dionex UltiMate3000 HPLC system coupled with a 3-angle miniDAWN TREOS static light scattering detector (Wyatt Technology). Sample volumes of 100 μL of APT1 WT and ATP2 WT at 40 μM were injected into a Superdex 75 10/300 GL column (GE Healthcare) previously equilibrated with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 2 mM TCEP at a flow rate of 0.5 mL/min. The data were further analyzed using ASTRA 6.1 software using the absorbance at 280 nm and the theoretical extinction coefficient for concentration measurements.
Ultimate 3000
Manufactured by Wyatt Technology
The Ultimate 3000 is a high-performance laboratory instrument designed for advanced analytical applications. It features cutting-edge technology and precision engineering to deliver reliable and accurate results. The core function of the Ultimate 3000 is to perform sophisticated analyses and measurements, enabling users to gather valuable data and insights for their research or testing needs.
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Lab products found in correlation
7 protocols using ultimate 3000
1
HPLC-SLS Analysis of Protein Complexes
2
Size Exclusion Chromatography and MALS Analysis
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Size exclusion chromatography and multiangle light scattering (SEC‐MALS) experiments were performed on a Dionex Ultimate 3000 HPLC system with an inline Wyatt miniDAWN TREOS MALS detector and Optilab T‐rEX refractive index detector. In addition, the elution profile of the protein was monitored with UV 280 attached to the Dionex system. For size exclusion chromatography, Superdex™ 200 Increase 10/300 GL column (GE Healthcare LifeSciences) was used. Fifty microliter of the purified UFL1/UFBP1 stored in buffer containing 25 mM Tris–pH 8.0, 300 mM NaCl and 2 mM DTT was loaded into the SEC column with Dionex autoloader at a concentration of 4 mg/ml and a flow rate of 0.3 ml/min was maintained throughout the experiment. Molar masses spanning elution peaks were calculated using ASTRA v6.0.0.108 (Wyatt).
3
Analyzing Phosphoprotein Interactions via SEC-MALS
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Size-exclusion chromatography and multiangle light scattering (SEC–MALS) experiments were performed on a Dionex Ultimate 3000 HPLC system with an inline Wyatt miniDAWN TREOS MALS detector and Optilab T-rEX refractive index detector. SEC–MALS experiments were performed on a Superdex S75 CL 10/300 (GE Healthcare) column in buffer containing 50 mM HEPES with 100 mM NaCl at pH 7.5. Phosphoubiquitin (140 μM) and phosphoparkin proteins (20 μM) were incubated for 30 min before injections. Molar masses spanning elution peaks were calculated using ASTRA v6.0.0.108 (Wyatt). For the size-exclusion analysis with UbcH7 or UbcH7∼Ub, phosphoparkin proteins (20 μM) were incubated with phosphoubiquitin (40 μM) and either UbcH7 (40 μM) or UbcH7∼Ub (40 μM) for 30 min prior to injections.
4
HPLC-SLS Analysis of Protein Complexes
5
SEC-MALS Analysis of Protein Samples
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Size exclusion chromatography and multi angle light scattering (SEC–MALS) experiments were performed on a Dionex Ultimate 3000 HPLC system with an inline Wyatt miniDAWN TREOS MALS detector and Optilab T-rEX refractive index detector. Initially SEC–MALS experiments were performed on a MAbPac SEC–1 (Dionex) column (Figure 1 ) and due to column degradation subsequent experiments were performed on a Superdex S200 CL 10/300 (GE Healthcare) column (Figures 5 and 7 ). Buffer conditions were 20 mM HEPES-KOH pH 7.5 with sodium chloride concentrations as stated in the text. Molar masses spanning elution peaks were calculated ASTRA v6.0.0.108 (Wyatt).
6
SEC-MALS Analysis of Protein
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The mass measurements were performed on a Dionex UltiMate3000 HPLC system coupled with a 3-angle miniDAWN TREOS static light scattering detector (Wyatt Technology). Sample volumes of 100 μl of APT1 WT and ATP2 WT at 40 μM were injected into a Superdex 75 10/300 GL column (GE Healthcare) previously equilibrated with 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 2 mM TCEP at a flow rate of 0.5 ml/min. The data were further analyzed using ASTRA 6.1 software using the absorbance at 280 nm and the theoretical extinction coefficient for concentration measurements.
7
SEC-MALS Analysis of UFL1/UFBP1 Protein
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Size exclusion chromatography and multi angle light scattering (SEC-MALS) experiments were performed on a Dionex Ultimate 3000 HPLC system with an inline Wyatt miniDAWN TREOS MALS detector and Optilab T-rEX refractive index detector.
In addition, the elution profile of the protein was monitored with UV 280 attached to the Dionex system. For size exclusion chromatography, Superdex 200 10/300 gl column (GE Healthcare LifeSciences) was used. 50 µl of the purified UFL1/UFBP1 stored in buffer containing 25 mM Tris pH 8.0, 300 mM NaCl, 2 mM DTT was loaded into the SEC column with Dionex auto loader at a concentration of 4 mg/ml and a flow rate of 0.3 ml/min was maintained throughout the experiment. Molar masses spanning elution peaks were calculated using ASTRA v6.0.0.108 (Wyatt).
In addition, the elution profile of the protein was monitored with UV 280 attached to the Dionex system. For size exclusion chromatography, Superdex 200 10/300 gl column (GE Healthcare LifeSciences) was used. 50 µl of the purified UFL1/UFBP1 stored in buffer containing 25 mM Tris pH 8.0, 300 mM NaCl, 2 mM DTT was loaded into the SEC column with Dionex auto loader at a concentration of 4 mg/ml and a flow rate of 0.3 ml/min was maintained throughout the experiment. Molar masses spanning elution peaks were calculated using ASTRA v6.0.0.108 (Wyatt).
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