Plectasin stock solutions were dialyzed against 10 mM acetate buffer pH 4.5 as described above, filtered (0.02 µm) and the concentration was measured. A protein stock solution of 20 mg/mL (4.5 mM) was obtained by dilution with the filtered dialysis buffer (0.02 µm). The respective formulations were obtained by a 10 times dilution in phosphate buffer with the desired pH. We found a final concentration of 2 mg/mL (450 µM) to give a good signal to noise ratio in DLS. The measurement was performed with a DynaPro® Plate ReaderTM II (Wyatt Technology) using Aurora 384 LV/EB plates (Brookes Life Science Systems). All measurements were performed at 25 °C with 5 s acquisition time and 20 acquisitions per well. All formulations were measured in triplicates. The analysis was performed DYNAMICS and final graphs were made with Origin® 2019(OriginLabs) and GraphPad Prism.
Dynapro plate reader 2
Manufactured by Wyatt Technology
Sourced in United States
The DynaPro Plate Reader II is a high-performance dynamic light scattering (DLS) instrument designed for measuring the size and distribution of particles in solution. It is capable of analyzing samples in microplates, cuvettes, or flow cells, and provides accurate and reliable data on the hydrodynamic size of macromolecules, proteins, nanoparticles, and other colloidal systems.
Automatically generated - may contain errors
Lab products found in correlation
Dynamics software, by Wyatt Technology (10 mentions)
Nanodrop, by Thermo Fisher Scientific (8 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
20 kd dialysis cassette, by Thermo Fisher Scientific (4 mentions)
Silicone oil, by Merck Group (3 mentions)
100kd cassette, by Thermo Fisher Scientific (2 mentions)
384 well plate, by Greiner (2 mentions)
High purity paraffin oil, by Merck Group (2 mentions)
Dynamics software version 7, by Wyatt Technology (2 mentions)
Dynamics software v7, by Wyatt Technology (2 mentions)
Dynamics v7, by Wyatt Technology (2 mentions)
Zetasizer nano z, by Malvern Panalytical (2 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Colorimetric assay, by Merck Group (1 mentions)
Fetuin a, by R&D Systems (1 mentions)
Nephelostar nephelometer, by BMG Labtech (1 mentions)
Whatman filter paper, by Cytiva (1 mentions)
Freedom evo, by Tecan (1 mentions)
H 7000 microscope, by Hitachi (1 mentions)
M bius instrument, by Wyatt Technology (1 mentions)
103 protocols using dynapro plate reader 2
1
Plectasin Solution Characterization by DLS
2
Characterization of Plectasin Formulations
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All plectasin samples were dialyzed in 10 mM Acetate pH 4.5 as described above, filtered (0.02 µm) and the concentration was measured. A protein stock solution of 20 mg/ml was obtained by dilution with the filtered dialysis buffer (0.02 µm). The respective formulations were obtained by a 10 times dilution. The measurement was performed with a DynaPro® Plate Reader TM II (Wyatt Technology) using Aurora 384 LV/EB plates (Brookes Life Science Systems). Silicone oil (Sigma Aldrich) was used for sealing the wells. All measurements were performed isothermally at 25°C with 5 s acquisition time and 20 acquisitions per well. All formulations were measured in technical triplicates. t0 measurement was performed at room temperature on three independent samples (biological triplicates), before one was taken for the stress study. The cumulant fit was used for analysis. Analysis was performed with DYNAMICS version 7.8.1.3 and final graphs were made with Origin® 2019 (OriginLabs).
3
Characterization of IFNα-2a Formulations
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IFNα-2a samples were concentrated to a concentration of approx. 20 mg/ml and dialyzed in 10 mM His pH 5.5, 10 mM His pH 7, and 10 mM Tris pH 8.5 as described above, filtered (0.22 µm) and the concentration was measured using a NanoDrop™ 8000 Spectrophotometer (Thermo Fisher). A protein stock solution of 20 mg/ml was obtained by dilution with the filtered dialysis buffer (0.2 µm). The respective formulations were obtained by a 20 times dilution. The measurement was performed with a DynaPro® Plate Reader TM II (Wyatt Technology) using Aurora 384 LV/EB plates (Brookes Life Science Systems). Silicone oil (Sigma Aldrich) was used for sealing the wells. All measurements were performed isothermally at 25°C with 5 s acquisition time and 20 acquisitions per well. All formulations were measured in technical triplicates. Analysis was performed using DYNAMICS version 7.8.1.3 and final graphs were prepared using Origin® 2019 (OriginLabs).
4
Plant-produced Humanized Anti-CD52 Antibody
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The human anti-CD52g mAb, HCA, was produced in Nicotiana benthamiana by Mapp Biopharmaceutical Inc. (San Diego, CA, USA). Genes containing the variable region sequences of H6-3C4 [16 (link), 19 (link)] were synthesized by Life Technologies (San Diego, CA, USA) and cloned into TMV and PVX plant expression vectors [27] (link) containing codon-optimized human lambda and human IgG1 constant regions. The vectors were then transformed into Agrobacterium tumefaciens strain ICF320 (Icon Genetics; Halle/Saale, Germany) which was used to transfect 4-week old Nicotiana plants by vacuum infiltration as previously described [22] (link). Seven days post-infiltration, antibody was extracted from the leaf tissue and purified by Protein A chromatography as previously described [22 (link), 28 (link)]. The Nicotiana plants used for this study were a transgenic strain in which fucosyl- and xylosyl- transferases were knocked down with RNAi so that the antibodies they produced had a humanized glycosylation pattern [21 (link), 29 (link)]. HCA QC was performed using multiple instruments/assays including the LabChip GXII Touch protein characterization system (quantification, purity, molecular weight sizing), SEC-HPLC, BioRad CFX real time PCR detection (thermal melt), and a Wyatt DynaPro Plate Reader II (DLS).
5
Monitoring Protein-Protein Interactions by DLS
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The interactions between hPXR LBD and hCAR LBD and that between hCAR LBD and RXRα LBD were monitored by dynamic light scattering (DLS), using a DynaPro Plate Reader II (Wyatt Technologies, Santa Barbara, CA). Additional details are indicated in the instrument manual (DYNAMICS User's Guide [M1400 Rev. K] (https://wyatt.com/files/literature/app-notes/dls-cuvette/ph-effects-stability ). Assays were performed using purified hCAR-LBD, RXRα LBD and hPXR-LBD at a concentration of 12 μM in 25 mM HEPES, pH 7.9, containing 150 mM NaCl, 5% glycerol, and 5 mM DTT. Measurements were performed on Dynamics using these parameters: Isotemp mode set at 25°C; data collection in DLS mode; DLS acquisition time: 10 s; read intervals: 1; number of acquisitions: 10; and auto-attenuation mode enabled. Signals from the samples were measured using an Aurora 384-well plate (Wyatt Technologies) after the samples had been incubated at room temperature for 1 h. Peptide ‘P’ (AKLLGLLAELRSINEA, an hCAR peptide corresponding to the hCAR heterodimer interface region) and peptide ‘C’ (AD LLGLLAK LD SINK A, in which residues with negative charges in peptide P were replaced with residues with positive charges) were used at a concentration of 120 μM. Data were analyzed using Dynamics 7.8 software and plotted with OriginPro.
6
Colloidal Particle Detection and CAC Determination
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A filtered 50 mM KPi buffer with a pH of 7.0 was used to create the samples, and the final DMSO concentration was set at 1% (v/v). The detection of colloidal particle generation was carried out using DynaPro Plate Reader II (Wyatt Technologies, Promega, Madison, WI, USA). Each substance was examined three times at 31.6 µM. The 8-point half-log dilutions of the compounds were performed in triplicate if colloids were discovered by DLS. To determine the critical aggregation concentration (CAC), data for each compound were spilt into two data sets based on aggregating (i.e., >106 scattering intensity) and non-aggregating (i.e., <106 scattering intensity) and fitted with separate nonlinear regression curves, and the point of intersection was determined using GraphPad Prism software version 9.1.1 (San Diego, CA, USA).
7
Measuring Serum Biomarkers in Mineralization
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Serum T50 was measured by Calciscon AG, Biel, Switzerland, as previously described using a Nephelostar nephelometer (BMG Labtech, Ortenberg, Germany)40 (link). The mean interassay CVA for T50 was 3.4%. CPP-II hydrodynamic radius was measured by dynamic light scattering using a DynaPro Plate Reader II (Wyatt Technology, Santa Barbara, CA, USA) as described by Chen et al.47 (link) The interassay CVA for CPP-II size was 4%. Commercial immunoassays were used to measure iFGF23 (Kainos Laboratories, Tokyo, Japan), 1,25 dihydroxyvitamin D Immunodiagnostic Systems, Boldon, UK), and fetuin-A (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. Mean interassay CVA were 3.8%, 5.5%, and 3.2%, respectively. Serum citrate was measured using a colorimetric assay (Sigma-Aldrich, Darmstadt, Germany) with a mean interassay CVA of 3.5%.
8
Fabrication of Stereocomplex Nanoparticles
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Stereocomplex nanoparticles, as shown in Fig. 1 , were fabricated as previously described.37 (link) In brief, oxygen-sensing polymer ( ) (45 mg) and mPEG-PDLA (45 mg) were dissolved in DMF (9 ml). The solution was briefly heated to facilitate dissolution of the polymers. With a syringe pump, the DMF solution was added to deionized (DI) water (81 ml) at a constant rate of . The solution was filtered through Whatman filter paper, and DMF was removed by dialysis (Select/Por; 12 to 14 kDa molecular weight cutoff; 2-l beaker filled with DI water; water changed every 2 h for 6 h, then allowed to stand overnight). The nanoparticle solution was then passed through a Whatman 200-nm Anotop filter. Nanoparticle sizes and polydispersities were analyzed via dynamic light scattering (DLS, Wyatt, DynaPro Plate Reader II), and results were consistent with previous reports (Fig. S5).25 (link)
9
Characterization of IFNα-2a Protein Formulations
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IFNα-2a samples were concentrated
by centrifugation to a concentration of approximately 20 mg/mL and
dialyzed in 10 mM His pH 5.5, 10 mM His pH 7, and 10 mM Tris pH 8.5
as described above. The material was filtered (0.22 μm), and
the concentration was measured using a NanoDrop 8000 spectrophotometer
(Thermo Fisher). A protein stock solution of 20 mg/mL was obtained
by dilution with the filtered dialysis buffer (0.2 μm). The
respective formulations were obtained by a 20 times dilution. The
measurement was performed with a DynaPro Plate Reader II (Wyatt Technology)
using Aurora 384 LV/EB plates (Brookes Life Science Systems). Silicone
oil (Sigma-Aldrich) was used for sealing the wells. All measurements
were performed isothermally at 25 °C with 5 s acquisition time
and 20 acquisitions per well. All formulations were measured in technical
triplicates. Analysis was performed using DYNAMICS version 7.8.1.3.
by centrifugation to a concentration of approximately 20 mg/mL and
dialyzed in 10 mM His pH 5.5, 10 mM His pH 7, and 10 mM Tris pH 8.5
as described above. The material was filtered (0.22 μm), and
the concentration was measured using a NanoDrop 8000 spectrophotometer
(Thermo Fisher). A protein stock solution of 20 mg/mL was obtained
by dilution with the filtered dialysis buffer (0.2 μm). The
respective formulations were obtained by a 20 times dilution. The
measurement was performed with a DynaPro Plate Reader II (Wyatt Technology)
using Aurora 384 LV/EB plates (Brookes Life Science Systems). Silicone
oil (Sigma-Aldrich) was used for sealing the wells. All measurements
were performed isothermally at 25 °C with 5 s acquisition time
and 20 acquisitions per well. All formulations were measured in technical
triplicates. Analysis was performed using DYNAMICS version 7.8.1.3.
10
Dynamic Light Scattering Protein Analysis
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DLS experiments were performed on a DynaPro-PlateReader II (Wyatt Technologies Corporation). Measurements of N samples in triplicates (1 mg/mL) were obtained at 25 °C and analyzed using Dynamics software (Wyatt).
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