Aliquots of MalFGK2-NSPr were analyzed by static light scattering. Static light scattering analysis were performed using a WTC-050S5 column (Wyatt Technologies) connected to a miniDAWN light scattering detector and interferometry refractometer (Wyatt Technologies). Data were recorded in real time and the molecular masses were calculated using the Debye fit method using the ASTRA software (Wyatt Technology).
Minidawn light scattering detector
Manufactured by Wyatt Technology
Sourced in Canada
The MiniDAWN is a light scattering detector designed to measure the molar mass and size of macromolecules and nanoparticles in solution. It utilizes multi-angle light scattering technology to provide accurate and reliable data on the molecular weight and radius of gyration of the sample.
Automatically generated - may contain errors
Lab products found in correlation
Astra software, by Wyatt Technology (3 mentions)
Optilab refractive index detector, by Wyatt Technology (2 mentions)
1260 infinity 2 hplc system, by Agilent Technologies (2 mentions)
Wtc 050s5 column, by Wyatt Technology (1 mentions)
Superdex 200 increase column 10 300 gl, by Wyatt Technology (1 mentions)
Astra 8, by Wyatt Technology (1 mentions)
Superose 6 10 30, by GE Healthcare (1 mentions)
Superdex s200 10 30, by GE Healthcare (1 mentions)
Superdex s75 10 30, by GE Healthcare (1 mentions)
Lc 20ad liquid chromatography system, by Shimadzu (1 mentions)
Superdex 200 increase 10 300 gl gel filtration column, by Cytiva (1 mentions)
Astra software package, by Wyatt Technology (1 mentions)
Coomassie plus the better bradford assay reagent, by Thermo Fisher Scientific (1 mentions)
1260 infinity 2 hplc system, by Wyatt Technology (1 mentions)
Superdex 200 increase 10 300 gl column, by Cytiva (1 mentions)
Akta pure fplc, by Cytiva (1 mentions)
Optilab differential refractive index detector, by Wyatt Technology (1 mentions)
11 protocols using minidawn light scattering detector
1
Molecular Mass Analysis by SLS
2
SEC-MALLS Analysis of Protein Samples
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Size exclusion chromatography multiple angle laser light scattering (SEC-MALLS) analysis was performed using a SuperoseTM Peptide column attached to the AKTA HPLC system at a flow rate of 0.5 mL/min in 20 mM Tris.HCl, pH 8.0, 150 mM NaCl, 1 mM DTT). A protein sample of 200 μM was used. The size exclusion chromatography column was followed in-line by a miniDAWN light scattering detector and an interometric refractometer (Wyatt Technologies, Santa Barbara, CA). Light scattering analysis was performed using a 690 nm wavelength laser. Voltage and light scattering intensity were calibrated with toluene yielding a constant of 8.534 × 10−6 for this study. A refractive index increment (dn/dc) estimate of 0.19 mL/g was used for protein concentration determination58 (link) and data were analysed using ASTRA software (Wyatt Technologies).
3
Molecular Weight Determination of hMLC1
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The molecular weight of the hMLC1 oligomer was determined using a Superdex-200 Increase 10/300 GL column coupled with a miniDAWN light scattering detector and an Optilab refractive index detector (Wyatt Technology). Purified hMLC1 proteins at a concentration of 1 mg ml−1 in 200 µl were loaded onto the column equilibrated with 150 mM NaCl, 20 mM HEPES, pH 7.5 and 2 mM DDM. Bovine serum albumin, CLC-ec1 and CLC-ec2 were used as the molecular weight standards. The data were analysed using ASTRA 8 software (Wyatt Technology).
4
Molecular Weight Analysis by SEC-MALLS
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Multi-angle laser light scattering experiments were performed using a miniDawn light scattering detector (Wyatt Technologies) in line with either a Superdex S75 10/30 (ASXL1DEU), Superose 6 10/30(BAP1) or Superdex S200 10/30(BAP1/ASXL1DEU) SEC column (GE Healthcare). SEC-MALLS runs were performed in 10 mM HEPES pH 7.5, 150 mM NaCl and 0.5 mM TCEP at 4 °C. Molecular weights were calculated by using the refractive index signal with the Astra software (Wyatt Technologies).
5
SEC-MALS Characterization of Protein Complexes
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SEC-MALS was conducted using a miniDAWN light scattering detector (Wyatt Technology Corporation, Santa Barbara, CA, USA) downstream of a LC-20AD liquid chromatography system (SHIMADZU, Kyoto, Japan) equipped with a Superdex 200 Increase 10/300 GL gel filtration column (Cytiva). The differential refractive index (SHOKO Science, Yokohama, Japan) downstream of MALS was used to estimate the protein concentration. The running buffer was phosphate-buffered saline (pH 7.4). Twenty microliters of the sample solution containing 0.1 mg of S-BD2, L-BD2, or BD1-L-BD2 were injected into the SEC column, and the protein was eluted at a flow rate of 0.4 mL/min. The data were analyzed using ASTRA version 8.0.1 (Wyatt Technology Corporation).
6
SEC-MALS Characterization of Biomolecules
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SEC-MALS
was performed using a 1260 Infinity II HPLC system (sampler,
pump, and UV–vis detector; Agilent Technologies, Santa Barbara,
CA) equipped with a miniDAWN light scattering detector and Optilab
refractive index detector (Wyatt Technologies, Santa Barbara, CA).
An AdvanceBio SEC 300 Å 2.7 μm, 4.6 × 300 mm column
(Agilent, Santa Clara, CA) and 150 mM sodium phosphate, pH 7.0, as
the mobile phase eluting at a rate of 0.2 mL min–1 and run time of 20 min were used for each experiment. For each run,
10 μL of sample with a MSNA concentration of 1 mg mL–1 in Milli-Q water was loaded onto the pre-equilibrated column. The
refractive index was used for the molecular weight calculations using
an average refractive index increment (dn/dc) of 0.1703 mL/g.
was performed using a 1260 Infinity II HPLC system (sampler,
pump, and UV–vis detector; Agilent Technologies, Santa Barbara,
CA) equipped with a miniDAWN light scattering detector and Optilab
refractive index detector (Wyatt Technologies, Santa Barbara, CA).
An AdvanceBio SEC 300 Å 2.7 μm, 4.6 × 300 mm column
(Agilent, Santa Clara, CA) and 150 mM sodium phosphate, pH 7.0, as
the mobile phase eluting at a rate of 0.2 mL min–1 and run time of 20 min were used for each experiment. For each run,
10 μL of sample with a MSNA concentration of 1 mg mL–1 in Milli-Q water was loaded onto the pre-equilibrated column. The
refractive index was used for the molecular weight calculations using
an average refractive index increment (dn/dc) of 0.1703 mL/g.
7
Characterization of Purified MtSEO-F1 Protein
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The concentration of purified soluble MtSEO‐F1 protein was determined by Bradford assay36 using Coomassie Plus—The Better Bradford Assay reagent (Thermo Fisher Scientific). We then fractionated 1 μg of protein by SDS‐PAGE on a 10% resolving gel as previously described.8 For immunodetection, the protein was blotted onto a nitrocellulose membrane4 and detected using an MtSEO‐F1‐specific antibody.37 MALS was carried out at 4°C on a miniDAWN light‐scattering detector (Wyatt Technology, Santa Barbara, CA) coupled to a Shodex RI detector online. Protein samples (20‐μl aliquots, 8.2 mg/ml) were loaded onto a Superdex S200 10/30 column (Thermo Fisher Scientific) equilibrated with 20 mM Tris (pH 8.0), 100 mM NaCl and 5% glycerol. Light scattering data were analyzed using the ASTRA software package (Wyatt Technology).
8
Protein Size Characterization by SEC-MALS
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One hundred microliters of CW1 protein samples were applied to a Superdex In 75 10/300 GL size-exclusion column (Cytiva) at concentrations between 10 and 200 µM. Size-exclusion chromatography was performed with a 1260 Infinity II HPLC system (Agilent) and a miniDawn light scattering detector (Wyatt) at a flow rate of 0.8 ml/min in 20 mM sodium acetate buffer, 100 mM NaCl, pH 5.3. Protein elution was detected by UV absorption at 280 nm and changes in the refractive index.
9
Characterization of tGemC1 Dimer
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Multi-angle laser light scattering (MALLS) experiments were performed in a Superdex 75 HR 10/30 column attached to an ÄKTA FPLC and coupled to a miniDAWN light-scattering detector (Wyatt Technology) and a Dn-1000 differential refractive-index detector (WGE Dr Bures). 100 µl of purified tGemC1 dimer at a concentration of ∼2.0 mg ml−1 were injected onto the column. Data analysis was carried out with ASTRA using a dn/dc value of 0.185. Size-exclusion chromatography runs for tGemC1–tGeminin were performed in a Superdex 75 HR 10/30 column attached to an ÄKTApurifier.
10
SEC-MALS Analysis of Supernanoparticles
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SEC-MALS was performed using an
Agilent Technologies 1260 Infinity II HPLC system (sampler, pump,
and UV–vis detector) equipped with a Wyatt Technologies miniDAWN
light scattering detector and Wyatt Technologies Optilab refractive
index detector. An Agilent AdvanceBio SEC 300 Å 2.7 μm
4.6 × 300 mm column and 150 mM sodium phosphate, pH 7.0, as mobile
phase eluting at a rate of 0.2 mL min–1 and run
time of 20 min were used for each experiment. For each run, 4 μL
of sample with a SNA concentration of 1 mg mL–1 in
Milli-Q water was loaded onto the pre-equilibrated column. Detector
signals were aligned with a bovine serum albumin (BSA) standard, which
was analyzed prior to SNA samples. The RI and MALS signals were used
for the MW calculations using an average refractive index increment
(dn/dc) of 0.1703 mL/g.
Agilent Technologies 1260 Infinity II HPLC system (sampler, pump,
and UV–vis detector) equipped with a Wyatt Technologies miniDAWN
light scattering detector and Wyatt Technologies Optilab refractive
index detector. An Agilent AdvanceBio SEC 300 Å 2.7 μm
4.6 × 300 mm column and 150 mM sodium phosphate, pH 7.0, as mobile
phase eluting at a rate of 0.2 mL min–1 and run
time of 20 min were used for each experiment. For each run, 4 μL
of sample with a SNA concentration of 1 mg mL–1 in
Milli-Q water was loaded onto the pre-equilibrated column. Detector
signals were aligned with a bovine serum albumin (BSA) standard, which
was analyzed prior to SNA samples. The RI and MALS signals were used
for the MW calculations using an average refractive index increment
(dn/dc) of 0.1703 mL/g.
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