The purified PML RING1–119 and mutants were subjected to gel filtration analysis (S100 column, GE Healthcare). The elution peaks, as monitored by UV absorption at 280 nm, were pooled separately and chosen for size exclusion chromatography-multi-angle light scattering ( SEC-MALS) characterization, respectively. In brief, the purified protein samples were concentrated and analyzed using a WTC-015S5 sized exclusion column (Wyatt Technology) which was connected to a 1260 infinity liquid chromatography system (Agilent Technology) equipped with inline DAWN HELEOS-II MALS and Optilab rEX differential refractive index detectors (Wyatt Technology). For each sample, a 40 μl injection volume and 0.5 ml min−1 flow rate were applied. Data were recorded and processed using ASTRA VI software (Wyatt Technology).
Dawn heleos 2 mals
Manufactured by Wyatt Technology
Sourced in United States
The Dawn HELEOS II MALS is a multi-angle light scattering (MALS) detector that measures the absolute molar mass and size of macromolecules and nanoparticles in solution. It is designed to be used in conjunction with other analytical techniques, such as size-exclusion chromatography or field-flow fractionation, to provide comprehensive characterization of complex samples.
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Lab products found in correlation
Optilab t rex, by Wyatt Technology (5 mentions)
Astra version 6, by Wyatt Technology (4 mentions)
Qels dsl, by Wyatt Technology (3 mentions)
Astra 6 software, by Wyatt Technology (2 mentions)
Wtc 015s5 sized exclusion column, by Wyatt Technology (2 mentions)
1260 infinity liquid chromatography system, by Agilent Technologies (2 mentions)
Aktamicro system, by GE Healthcare (2 mentions)
Astra 6, by Wyatt Technology (2 mentions)
Protein standard, by Bio-Rad (2 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (2 mentions)
Astra 7, by Wyatt Technology (2 mentions)
Sw3000 column, by Tosoh (2 mentions)
Optilab rex differential refractive index detector, by Wyatt Technology (1 mentions)
S100 column, by GE Healthcare (1 mentions)
Optilab rex, by Wyatt Technology (1 mentions)
Tskgel g3000swxl column, by Tosoh (1 mentions)
Astra6 software package, by Wyatt Technology (1 mentions)
Superdex 200 increase 10 300 gl column, by Cytiva (1 mentions)
Origin software, by OriginLab (1 mentions)
Astra software version 6, by Wyatt Technology (1 mentions)
16 protocols using dawn heleos 2 mals
1
SEC-MALS Analysis of PML RING Mutants
2
Gel Filtration Analysis of SafDAA-dsc and Mutants
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SafDAA-dsc and mutants were subjected to gel filtration analysis using a WTC-015S5 sized exclusion column (Wyatt Technology). The elution of each sample was analyzed by a 1260 infinity liquid chromatography system (Agilent Technology) linked with inline DAWN HELEOS-II MALS and Optilab rEX differential refractive index detectors (Wyatt Technology). For each run, a 40-μl sample (2.5 mg/ml) was injected. The sample was eluted at a flow-rate of 0.5 ml/min for SafDAA-dsc and mutants. Data were recorded and analyzed using ASTRA VI software (Wyatt Technology).
3
Size Exclusion HPLC Analysis of Protein Aggregates
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Aggregates were analyzed by size exclusion-HPLC (SE-HPLC) with light scattering (LS) detection (SE-HPLC-LS). SE-HPLC measurements were made on an Agilent 1100 HPLC system with a Tosoh Bioscience (Minato, Tokyo, Japan) TSK-GEL G3000SWxl column (5 µm, 7.8 × 300 mm). The SE-HPLC system was equipped with an LS detector (DAWN® HELEOS™ II MALS; Wyatt Technology Corporation, Goleta, CA, USA), and a refractive index (RI) detector (Wyatt Optilab rEX RI; Wyatt Technology Corporation). Samples without dilution were injected into the HPLC system, and the levels of high molecular weight (HMW) species and monomer were quantified and their molecular weights calculated. A differential RI value of 0.185 mL/g was used for molecular weight calculation.
4
Characterization of VCPKMT-p97-N/D1 complex
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Individual purified VCPKMT and p97-N/D1 (N21-Q458) were mixed at 2:1 ratio, ensuring an excess amount of VCPKMT was presented. The mixture was then incubated at 277 K for two hours before being subjecting to size-exclusion chromatography column. A total of 100 μl of protein solution was loaded onto a Superdex 200 Increase 10/300 GL column (Cytiva, USA), which has been pre-equilibrated with a buffer containing 20mM Tris-HCl, 0.1mM TCEP, and 200mM NaCl. The elution of the protein was carried out at a flow rate of 0.5 ml/min and was subsequently applied to inline DAWN Heleos II MALS and Optilab T-Rex differential refractive index detector (Wyatt Technology, USA). The resulting data were analyzed using the ASTRA 6 software package (Wyatt Technology, USA) and the final results were visualized using Origin software (OriginLab, USA).
5
Size-exclusion Chromatography Protein Analysis
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Size-exclusion chromatography experiments employed a Superose 6 10/300 GL column (GE Healthcare) equilibrated in buffer B. The column was calibrated with thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.4 kDa). Experimental proteins were loaded at 4 mg ml−1 in buffer B and molecular masses were determined using a DAWN HELEOS II MALS with an on-line Optilab T-rEX interferometric refractometer (Wyatt Technology). The refractive index increment (dn/dc) was estimated as 0.185 and the molecular mass of each protein within defined chromatographic peaks was calculated using ASTRA software version 6.0 (Wyatt Technology) as recommended by the manufacturer.
6
SEC-MALS Analysis of Protein Complexes
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For SEC-MALS analyses 100 μL protein complex at 2 mg⋅ml−1 was passed over a Superdex 200 10/300 Increase GL column (GE Healthcare). The column output was fed into a DAWN HELEOS II MALS, followed by an Optilab T-rEX differential refractometer (Wyatt Technology); full methodology is provided in SI Appendix .
7
Structural Analysis of DNA Repair Complex
8
GMMA Size Characterization by HPLC-SEC-MALS
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GMMA samples were analyzed by HPLC-SEC
with Tosoh TSK gel G6000PW (30 cm × 7.5 mm) + G4000PW (30 cm
× 7.5 mm) columns in series equilibrated in PBS (PBS tablets,
Medicago) and with in-line UV, fluorescence emission, and MALS detectors.
A Wyatt Dawn Heleos II MALS equipped with fused silica cell and a
660 nm laser source were used. A volume of 80 μL of samples
with concentrations of 100 μg/mL protein content were injected
and eluted with a flow rate of 0.5 mL/min (run time 70 min). All of
the dilutions were made in PBS. MALS data were collected using ASTRA
6 software (Wyatt Technology) with “particles” template
and analyzed using “Sphere” model. The size of GMMA
was expressed by the number average geometric radius Rn, weight average geometric radius Rw, and Z-average geometric radius Rz values.
with Tosoh TSK gel G6000PW (30 cm × 7.5 mm) + G4000PW (30 cm
× 7.5 mm) columns in series equilibrated in PBS (PBS tablets,
Medicago) and with in-line UV, fluorescence emission, and MALS detectors.
A Wyatt Dawn Heleos II MALS equipped with fused silica cell and a
660 nm laser source were used. A volume of 80 μL of samples
with concentrations of 100 μg/mL protein content were injected
and eluted with a flow rate of 0.5 mL/min (run time 70 min). All of
the dilutions were made in PBS. MALS data were collected using ASTRA
6 software (Wyatt Technology) with “particles” template
and analyzed using “Sphere” model. The size of GMMA
was expressed by the number average geometric radius Rn, weight average geometric radius Rw, and Z-average geometric radius Rz values.
9
Molecular Mass and Oligomerization of CAF-1 p150L
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SEC-MALS was used to determine the molecular mass and oligomerization status of CAF-1 p150L. Samples were injected onto a Superdex 200 Increase 10/300 GL column at 0.35 mL/min using an ÄKTAmicro FPLC (GE Healthcare). The running buffer contained 10 mM sodium phosphate, pH 7.2, 0.1 mM TCEP, and either 150 or 500 mM NaCl. Samples were passed through a Dawn HELEOS II MALS and OptiLab T-rEX online refractive index detectors (Wyatt Technology, Santa Barbara, CA, USA) after calibration with the BSA monomer. Data were processed with ASTRA Version 6.1.6.5 (Wyatt Technology, Santa Barbara, CA, USA).
10
Molecular Mass Determination by SEC-MALS
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Molecular masses were determined using a SEC column (Shodex KW-803) coupled with Dawn HELEOS II MALS and Optilab T-rEX Refractive Index Detector (Wyatt Technology). The system was equilibrated with PBS buffer supplemented with 1 mM DTT. A total of 50 μl of each sample was injected onto the SEC column at a flow rate of 0.5 ml/minute. Data were analyzed using ASTRA 6 software (Wyatt Technology). Bovine serum albumin was used to calibrate and normalize the detectors.
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