Size exclusion chromatography/multiangle light scattering analysis was performed on an OmniSEC system (Malvern Panalytical). Samples consisted of 0.2μm-filtered, 1 mg/ml purified proteins in 20 mM Tris pH 8, 150 mM NaCl, 25 mM imidazole buffer. Injection volumes were 50 μl. Samples were measured at 30 °C with a flow rate of 0.7 mL/min. SEC-MALS runs were performed by the BIC facility of CEITEC MU, Brno.
Omnisec system
Manufactured by Malvern Panalytical
Sourced in United Kingdom
The OMNISEC system is a multi-detector size exclusion chromatography (SEC) instrument designed for the comprehensive characterization of macromolecules and nanoparticles. The system incorporates multiple detectors, including a laser light scattering detector, a refractive index detector, and a viscometer, which work together to provide detailed information about the molecular weight, size, and structure of the analyzed samples.
Automatically generated - may contain errors
Lab products found in correlation
Tensor 27 spectrometer, by Bruker (1 mentions)
Synergy 2 multi mode microplate reader, by Agilent Technologies (1 mentions)
Avance 300, by Bruker (1 mentions)
Superdex 200 increase 10 300 sec column, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Polycal standards, by Malvern Panalytical (1 mentions)
Gpc 50, by Polymer Laboratories (1 mentions)
Superdex 200 increase 10 300, by GE Healthcare (1 mentions)
6 protocols using omnisec system
1
Protein Size Characterization by SEC-MALS
2
Multimodal Characterization of Polymers
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1H NMR spectra were recorded on 1.5% wt. solutions in deuterated chloroform and dimethyl sulfoxide (d6-DMSO) using a Bruker Avance 300 or 400 MHz Bruker spectrometer.
FT-IR spectra were recorded in ATR mode (Golden Gate) on a Tensor 27 Bruker spectrometer (Bruker UK Limited, UK) equipped with a 3000 Series TM High Stability Temperature Controller (Specac, UK).
Gel permeation chromatography (GPC) analysis was performed using triple detection conditions with a Malvern OmniSec System (Malvern, UK) comprising of a D6000M and D3000 column in sequence and operating online at 50 °C with HPLC grade DMF containing 0.1% LiBr as the mobile phase at a flow rate of 1.0 mL/min. Calibration was performed using a poly(methyl methacrylate) standard of a known molecular weight, intrinsic viscosity, and dn/dc.
UV-Vis and fluorescence readings were obtained through a BioTek Synergy 2 multimode microplate reader (BioTek, UK) at a temperature of 37 °C.
FT-IR spectra were recorded in ATR mode (Golden Gate) on a Tensor 27 Bruker spectrometer (Bruker UK Limited, UK) equipped with a 3000 Series TM High Stability Temperature Controller (Specac, UK).
Gel permeation chromatography (GPC) analysis was performed using triple detection conditions with a Malvern OmniSec System (Malvern, UK) comprising of a D6000M and D3000 column in sequence and operating online at 50 °C with HPLC grade DMF containing 0.1% LiBr as the mobile phase at a flow rate of 1.0 mL/min. Calibration was performed using a poly(methyl methacrylate) standard of a known molecular weight, intrinsic viscosity, and dn/dc.
UV-Vis and fluorescence readings were obtained through a BioTek Synergy 2 multimode microplate reader (BioTek, UK) at a temperature of 37 °C.
3
Size Exclusion Chromatography of Proteins
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SEC coupled to RALS was performed on an OMNISEC system (Malvern Instruments, Worcestershire, UK) using a Superdex 200 Increase 10/300 SEC column (GE Healthcare). Prior to each run, the system was extensively equilibrated in running buffer (20 mM Tris, pH 7.5, 150 mM KCl). Protein concentrations were determined from online refractive index (RI) measurements assuming a dn/dc of 0.185 mL/mg. 50 µL of a calibration standard (BSA, Sigma, at 3 mg/mL) and samples (at 4 mg/mL) were injected twice. All experiments were conducted at 20 °C with a flow rate of 0.5 mL/min for 70 min. Molar masses were calculated as described by the manufacturer using OMNISEC v10 software.
4
Comprehensive GPC/SEC Analysis of PLA
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Gel
permeation chromatography (GPC)/size-exclusion chromatography
(SEC) was performed on an integrated OMNISEC system (Malvern PANalytical
Ltd., UK) equipped with a D6000M and a D4000 column (10 and 6 μm
particle sizes, respectively, 300 × 8 mm) using a triple-detection
method (refractive index, viscometer, and dual-angle light scattering
detector at 7 and 90°). Tetrahydrofuran (THF) stabilized with
250 ppm butylated hydroxytoluene was used as the eluent at a temperature
of 35 °C and a flow rate of 1.0 mL/min. The system was calibrated
with the polystyrene (PolyCAL Standards, Malvern PANalytical Ltd.,
UK) 105 kDa narrow standard and verified with a 250 kDa broad standard
of known dispersity, intrinsic viscosity, and dn/dc. Data analysis was performed using OMNISEC software V11.10.
Prior to each analysis, samples were dissolved in THF at a known concentration
and room temperature (RT), and the resulting solutions were filtered
through a 0.22 μm poly(tetrafluoroethylene) (PTFE) filter. Triple
detection was used to obtain absolute MW distributions, intrinsic
viscosity, hydrodynamic radius (RH), and
Mark–Houwink parameters (log K and a) of the PLAs. The radius of gyration Rg was not calculated as the synthesized PLAs have too
low dn/dc (dn/dc data are shown in the Supporting Information) and hydrodynamic
size to produce anisotropic scattering, which is required for Rg calculations.
permeation chromatography (GPC)/size-exclusion chromatography
(SEC) was performed on an integrated OMNISEC system (Malvern PANalytical
Ltd., UK) equipped with a D6000M and a D4000 column (10 and 6 μm
particle sizes, respectively, 300 × 8 mm) using a triple-detection
method (refractive index, viscometer, and dual-angle light scattering
detector at 7 and 90°). Tetrahydrofuran (THF) stabilized with
250 ppm butylated hydroxytoluene was used as the eluent at a temperature
of 35 °C and a flow rate of 1.0 mL/min. The system was calibrated
with the polystyrene (PolyCAL Standards, Malvern PANalytical Ltd.,
UK) 105 kDa narrow standard and verified with a 250 kDa broad standard
of known dispersity, intrinsic viscosity, and dn/dc. Data analysis was performed using OMNISEC software V11.10.
Prior to each analysis, samples were dissolved in THF at a known concentration
and room temperature (RT), and the resulting solutions were filtered
through a 0.22 μm poly(tetrafluoroethylene) (PTFE) filter. Triple
detection was used to obtain absolute MW distributions, intrinsic
viscosity, hydrodynamic radius (RH), and
Mark–Houwink parameters (log K and a) of the PLAs. The radius of gyration Rg was not calculated as the synthesized PLAs have too
low dn/dc (dn/dc data are shown in the Supporting Information) and hydrodynamic
size to produce anisotropic scattering, which is required for Rg calculations.
5
Polymer Characterization via GPC
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The analysis was
performed using triple detection conditions with either a GPC50 (Polymer
Laboratories, UK; THF mobile phase) or a Malvern OmniSec System (Malvern,
UK; DMF mobile phase). Molecular weight and molecular weight distribution
of PPS polymers were determined using a PLgel 5 μm MIXED-D column
in series with a PLgel 3 μm MIXED-E operating online at 30 °C.
THF was used as an eluent at a flow rate of 1.0 mL/min. Two linear
polystyrene standards (Malvern, UK) for calibration (narrow) and for
verification (broad) were used for the analysis of the polymers. Block
copolymers of PPS with polyacrylamides were analyzed using a D3000
column operating online at 50 °C with HPLC grade DMF containing
0.1% LiBr as the mobile phase at a flow rate of 0.8 mL/min. Calibration
was performed using a poly(methyl methacrylate) standard.
performed using triple detection conditions with either a GPC50 (Polymer
Laboratories, UK; THF mobile phase) or a Malvern OmniSec System (Malvern,
UK; DMF mobile phase). Molecular weight and molecular weight distribution
of PPS polymers were determined using a PLgel 5 μm MIXED-D column
in series with a PLgel 3 μm MIXED-E operating online at 30 °C.
THF was used as an eluent at a flow rate of 1.0 mL/min. Two linear
polystyrene standards (Malvern, UK) for calibration (narrow) and for
verification (broad) were used for the analysis of the polymers. Block
copolymers of PPS with polyacrylamides were analyzed using a D3000
column operating online at 50 °C with HPLC grade DMF containing
0.1% LiBr as the mobile phase at a flow rate of 0.8 mL/min. Calibration
was performed using a poly(methyl methacrylate) standard.
6
Determining Oligomeric State of DS-epi1 via SEC-MALS
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To compare the mono- or multimeric state of DS-epi1 at different protein concentrations, proteins were separated on an AdvanceBio SEC UHPLC column (300 Å, 2.7 μm, 4.6 × 300 mm, Agilent) with a mobile phase consisting of HEPES (20 mM), NaCl (150 mM) and MnCl2 (5 mM).
For absolute molecular weight determination, multi-detection SEC was performed using a Malvern Panalytical OMNISEC system (Malvern, UK) consisting of Refractive Index (RI), right angle and low angle light scattering (RALS/LALS) and differential viscometer. For chromatographic separation, a Superdex 200 increase 10/300 (GE Life Sciences) was used with a mobile phase consisting of HEPES (20 mM) and NaCl (150 mM). A dn/dc of 0.185 ml g–1 was used to process the protein samples. All data was collected and processed using OMNISEC v10.
For absolute molecular weight determination, multi-detection SEC was performed using a Malvern Panalytical OMNISEC system (Malvern, UK) consisting of Refractive Index (RI), right angle and low angle light scattering (RALS/LALS) and differential viscometer. For chromatographic separation, a Superdex 200 increase 10/300 (GE Life Sciences) was used with a mobile phase consisting of HEPES (20 mM) and NaCl (150 mM). A dn/dc of 0.185 ml g–1 was used to process the protein samples. All data was collected and processed using OMNISEC v10.
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