A total of 100 μL of the PfAlu118 RNA, PfSRP9/14 heterodimer and PfSRP Alu complex at ~1 mg/mL were subjected to SEC using a Superdex 200 10/300 column (GE Healthcare) coupled to a MALS system (miniDAWN Tristar, Wyatt Technologies) and refractive index detector (RI-71, Shodex) at 4°C. The SEC column was pre-equilibrated with buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 10 mM KCl and 1 mM DTT, and the data were analyzed using Astra 6 software (Wyatt Technology).
Minidawn tristar
Manufactured by Wyatt Technology
Sourced in United States
The MiniDAWN Tristar is a multi-angle light scattering (MALS) detector designed for the analysis of macromolecules and nanoparticles. It measures the angular dependence of scattered light intensity, which provides information about the size, molar mass, and conformation of the analyzed samples.
Automatically generated - may contain errors
Lab products found in correlation
Optilab dsp, by Wyatt Technology (3 mentions)
Superdex 200 10 300 column, by GE Healthcare (2 mentions)
Astra 6, by Wyatt Technology (1 mentions)
Kta purifier 100, by GE Healthcare (1 mentions)
Prism 7, by GraphPad (1 mentions)
Superose 12 10 300 gl column, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
9 protocols using minidawn tristar
1
SEC Analysis of Pf Protein Complexes
2
Protein Characterization by MALLS
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Purified protein was run on a Superdex 200 gel filtration column (GE Healthcare) using GF buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM DTT) in line with a MiniDawn Tristar (Wyatt Technologies) Multi-Angle Laser Light Scattering (MALLS) detector, connected to a Shodex RI 101 (SHOWA DENKO K.K.) refractive index detector. Wyatt Technologies software (ASTRA) was used to determine the corresponding peaks’ molecular weight based on the refractive index.
3
Characterization of RNA Structural States
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Unfolded and folded RNA samples were obtained as described above and subjected to size-exclusion chromatography (SEC) using a Superdex™ 75 10/300 GL column equilibrated in a buffer containing 20-mM Tris/HCl pH 8.0, 200-mM NaCl, 10-mM KCl, 10-mM MgCl2. SEC was coupled to a MALS (miniDAWN Tristar, Wyatt Technologies) and refractive index detector (RI-71, Shodex) allowing determination of the absolute molar mass. For calculation of the molar mass, a dn/dc value of 0.170 was used.
4
Molecular Mass Determination by MALS
5
SEC-MALS Protein Characterization
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SEC-MALS measurements were performed on an ÄKTApurifier 100 (GE Healthcare) connected to a tri-angle detector MiniDAWN Tristar (Wyatt Technologies). For each experiment, a volume of 100 μl of 100 μm protein solution was injected into a Superdex 200 10/300 column (GE Healthcare), previously equilibrated with 20 mm MES, pH 6.8, 300 mm NaCl, and 1 mm TCEP. Molecular weights of main peaks were determined using the manufacturer's software (ASTRA) and assuming a specific refractive index increment (dn/dc) of 0.185 ml g−1. Chromatographic profiles and molecular weights were plotted using Prism 7 (GraphPad).
6
Molecular Mass Determination by MALS
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For molecular mass determination by MALS, protein samples were injected into a Superdex 200 (10/300 GL) gel filtration column equilibrated with the gel filtration buffer. The chromatography system was attached to a three-angle light scattering detector (miniDAWN TRISTAR) and a refractive index detector (Optilab DSP) (Wyatt Technology). Data were collected every 0.5 s with a flow rate of 0.5 ml/min. Data analysis was carried out using ASTRA V.
7
Molecular mass analysis of NLRP1
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After the Superdex 200 10/30 size-exclusion column was equilibrated with buffer (500 mM NaCl, 20 mM CHES-HCl pH 9.0), 500 µL of the samples at 0.5 mg/mL concentration were injected onto the mini-DAWN Tristar (Wyatt Technologies, USA) to analyze the molecular mass and oligomeric state of NLRP1CARD and mutants. Each protein at a flow rate of 0.5 mL/min was passed through UV detector, a refractometer, and a multi-angle laser light-scattering detector. The sample data were processed with the manufacturer’s software named ASTRA (Wyatt Tech). Relative weight-averaged molecular masses of samples were based on absorption coefficients from the calculated result of polypeptide sequence of proteins.
8
Size-Exclusion Chromatography and Light Scattering
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Proteins were subjected to size-exclusion chromatography in 20 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, pH 8.0, on a Superose 12 10/300 GL column (GE Healthcare). The light scattering properties of eluting species were measured in 3.125 μl slices using an on-line miniDAWN TriStar light scatterer-detector (Wyatt Technology). The refractive index relative to the eluent was measured using an OptiLab DSP interferometric refractometer, and the concentration of the eluting protein was estimated based on a refractive index increment of 0.19 ml g -1 . Data were processed and analysed on the ASTRA 4.90.07 software (Wyatt Technology).
9
Determining ABIN2-triUb Complex Mass
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The molecular mass of the ABIN2-triUb complex was determined by MALS. Protein samples were injected into a Superdex 200 10/300 GL gel-filtration column (GE Healthcare) equilibrated in a buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The chromatography system was coupled to a three-angle light-scattering detector (mini-DAWN TRISTAR) and a refractive index detector (Optilab DSP) (Wyatt Technology). Data were collected every 0.5 s with a flow rate of 0.2 mL/min. Data analysis was carried out using ASTRA V.
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