First, 60 µl of hTLR2 in PBS buffer (pH 7.4) was incubated at room temperature for 2 hours with indicated concentrations of CU-T12-9 or Pam3CSK4. Then, 60 µl of hTLRI in PBS buffer (pH 7.4) was added to the reaction mixture, which was incubated at 37°C for an additional 2 hours. The hTLRI and hTLR2 proteins were mixed in an equimolar ratio such that the final concentration of hTLR1 was 1 mg/ml (~10 µM) and hTLR2 was 0.8 mg/ml (~10 µM). All buffers and samples were filtered through a 0.1-µm filter. Then, 100 µl of prepared samples was analyzed by injection onto a Shodex KW80X size exclusion column running in PBS buffer (pH 7.4) with a flow rate of 1 ml/min. The column was in line with a multiangle light scattering detector (DAWN EOS, Wyatt Technologies), a refractive index detector (Optilab DSP, Wyatt Technologies), and an absorbance detector (UV 3000, Spectra System) for data collection. Data were analyzed with Astra 4.9 software (Wyatt Technologies).
Astra 4
Manufactured by Wyatt Technology
Sourced in United States
ASTRA 4.9 is a software package developed by Wyatt Technology for the analysis of light scattering data. It provides tools for the calculation of molecular weights, sizes, and other physical properties of macromolecules and nanoparticles.
Automatically generated - may contain errors
Lab products found in correlation
Optilab dsp interferometric refractometer, by Wyatt Technology (3 mentions)
Dawn eos, by Wyatt Technology (2 mentions)
Optilab dsp, by Wyatt Technology (2 mentions)
Superdex 200 increase column, by Cytiva (1 mentions)
Superdex 200 increase 5 150, by GE Healthcare (1 mentions)
Prism 8, by GraphPad (1 mentions)
Superdex 200 increase 5 150 gl column, by Cytiva (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Dawn dsp f, by Wyatt Technology (1 mentions)
Tskgel gmpwxl, by Tosoh (1 mentions)
Tskgel 2500 pwxl, by Tosoh (1 mentions)
7 protocols using astra 4
1
Size-Exclusion Analysis of TLR1/TLR2 Binding
2
hTLR1/hTLR2 Interaction Characterization
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First, 60 μl of hTLR2 in PBS buffer (pH 7.4) was incubated at room temperature for 2 hours with indicated concentrations of CU-T12-9 or Pam3CSK4. Then, 60 μl of hTLR1 in PBS buffer (pH 7.4) was added to the reaction mixture, which was incubated at 37°C for an additional 2 hours. The hTLR1 and hTLR2 proteins were mixed in an equimolar ratio such that the final concentration of hTLR1 was 1 mg/ml (~10 μM) and hTLR2 was 0.8 mg/ml (~10 μM). All buffers and samples were filtered through a 0.1-μm filter. Then, 100 μl of prepared samples was analyzed by injection onto a Shodex KW80X size exclusion column running in PBS buffer (pH 7.4) with a flow rate of 1 ml/min. The column was in line with a multiangle light scattering detector (DAWN EOS, Wyatt Technologies), a refractive index detector (Optilab DSP, Wyatt Technologies), and an absorbance detector (UV 3000, Spectra System) for data collection. Data were analyzed with Astra 4.9 software (Wyatt Technologies).
3
Molecular Weight Determination of NS1 mSPE-7 IgE
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The three peaks isolated from the S200 Increase HPLC purification of NS1 mSPE-7 IgE were run again on the Superdex™ 200 Increase column (Amersham Pharmacia Biotech) to determine their molecular weight, using an inline miniDAWN light-scattering detector and an Optilab DSP Interferometric Refractometer, and the data was analysed using the ASTRA 4.9 software (Wyatt Technology).
4
Characterization of IgM Mutants by MALLS
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MALLS studies were performed in-line with SEC on mutant IgM protein samples to assess mono-dispersity and the molecular mass of the protein samples. The peaks corresponding to the homodimeric Fcμ2-4 mutants from SEC were run on the Superdex 200 Increase 5/150 column (GE Healthcare) using an in-line miniDAWN multi-angle light scattering detector and an Optilab DSP Interferometric Refractometer (Wyatt Technology). The data were analyzed using the ASTRA 4.9 software (Wyatt Technology).
5
SEC-MALLS Analysis of Protein Complexes
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SEC-MALLS was performed using a Superdex 200 Increase 5/150 GL column (Cytiva) at a flow rate of 0.3 ml/min using a miniDAWN light-scattering detector and an Optilab DSP Interferometric Refractometer (both Wyatt Technology) at room temperature. Samples were injected at a volume of 50 μl and at the following concentrations: C1QTNF4-d1 at 3 mg/ml, GFP A206K at 2 mg/ml, GFP-d1 at 1.1 mg/ml, GFP-d2 at 1.3 mg/ml and GFP-C1QTNF4 at 1.1 mg/ml in 50 mM Tris, 150 mM NaCl, 4 mM CaCl2, 0.1% NaN3, pH 7.4; C1QTNF4 at 1.4 mg/ml in 50 mM Tris, 500 mM NaCl, 4 mM CaCl2, 0.1% NaN3, pH 8.55. The data were collected and analyzed using the ASTRA 4.9 software (Wyatt Technology). The data for refractive index and molecular mass were visualized by plotting in Prism 8 (GraphPad).
6
Starch Polysaccharide Molecular Weight Analysis
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A high-performance size exclusion chromatography (HPSEC) system with a multiangle laser light scattering (MALLS) detector and a differential refractive index detector (RI) were used to determine the molecular weight and radii of gyration of the starch polysaccharide molecules. The high-performance size exclusion chromatography (HPSEC) system consisted of a pump (Ultimate 3000, Dionex, Palo Alto, CA, USA), an injection valve (model 7021, Rheodyne, Palo Alto, CA, USA), a guard column (TSK PWH, Tosoh Corporation, Tokyo, Japan) and two connected size exclusion columns: TSKgel GMPWXL (300 mm × 7.8 mm, Tosoh Corporation, Tokyo, Japan) and TSKgel 2500 PWXL (300 mm × 7.8 mm, Tosoh Corporation, Tokyo, Japan). A multiangle laser light scattering (MALLS) detector (Dawn-DSP-F, Wyatt Technology, Santa Barbara, CA, USA) and a differential refractive index detector (model SE71, Shodex, Tokyo, Japan) were connected to the columns.
The flow rate of the mobile phase and the sample injection volume were 0.4 mL · min−1 and 500 μL, respectively. Calculation of the weight-average molecular weight (Mw) and radius of gyration (Rg) were performed using Astra 4.70 software (Wyatt Technology, Santa Barbara, CA, USA).
The flow rate of the mobile phase and the sample injection volume were 0.4 mL · min−1 and 500 μL, respectively. Calculation of the weight-average molecular weight (Mw) and radius of gyration (Rg) were performed using Astra 4.70 software (Wyatt Technology, Santa Barbara, CA, USA).
7
Determination of DNA Molecular Weight
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Determination of molecular weight and radii of gyration of DNA and their derivatives were measured by means of high-performance size exclusion chromatography (HPSEC) coupled with multiangle laser light-scattering (MALLS) detector and differential refractive index (RI) detector. The high-performance size exclusion chromatography system and methods of measurement are described in our previous publication (Nowak et al. 2012) .
Calculation of the weight-average molecular weight (M w ) and radius of gyration (R g ) used Astra 4.70 software (Wyatt Technology, Santa Barbara, California, USA). A Berry plot with third-order polynomial fit was applied for the calculation of M w and R g values (Hanselmann et al. 1996; Bello-Perez et al. 1996) .
Calculation of the weight-average molecular weight (M w ) and radius of gyration (R g ) used Astra 4.70 software (Wyatt Technology, Santa Barbara, California, USA). A Berry plot with third-order polynomial fit was applied for the calculation of M w and R g values (Hanselmann et al. 1996; Bello-Perez et al. 1996) .
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