Fifty microliters of JIP3LZ was injected onto a WTC-030S5 column, pre-equilibrated with buffer containing 25 mM HEPES pH 7.5, 0.1 M NaCl and 1 mM TCEP at a flow rate of 1 ml/min. Data were recorded using a DAWN 8+ multi-angle light scattering (LS) detector, an Optilab T-rEX differential refractive index (dRI) detector and UV absorbance (UV) detector (Wyatt Technology) and analyzed with the Astra 6.2 software package provided by the manufacturer (Wyatt Technology).
Optilab t rex differential refractive index detector
Manufactured by Wyatt Technology
Sourced in United States
The Optilab T-rEX differential refractive index detector is a laboratory instrument designed to measure changes in the refractive index of a sample. It is a core component used in various analytical techniques, such as size exclusion chromatography and light scattering analysis, to provide detailed information about the physical and chemical properties of samples.
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Lab products found in correlation
Polargel m columns, by Agilent Technologies (2 mentions)
Dawn heleos mals detector, by Wyatt Technology (2 mentions)
Dawn heleos 2 multi angle light scattering detector, by Wyatt Technology (2 mentions)
Astra 7, by Wyatt Technology (2 mentions)
Astra software package, by Wyatt Technology (1 mentions)
Model 1260, by Agilent Technologies (1 mentions)
Agilent 1260 autosampler, by Agilent Technologies (1 mentions)
Ultimate 3000 system, by Thermo Fisher Scientific (1 mentions)
Spd 20a uv vis detector, by Shimadzu (1 mentions)
Heleos 2 multi angle light scattering, by Wyatt Technology (1 mentions)
Superdex s200, by GE Healthcare (1 mentions)
Ultimate 3000 autosampler, by Thermo Fisher Scientific (1 mentions)
Polargel m guard column, by Agilent Technologies (1 mentions)
Dawn heleos 2 detector, by Wyatt Technology (1 mentions)
Superdex 200 10 300 column, by Cytiva (1 mentions)
Minidawn treos multi angle static light scatteringdetector, by Wyatt Technology (1 mentions)
1260 infinity hplc system, by Agilent Technologies (1 mentions)
Agilent 1100 series, by Agilent Technologies (1 mentions)
Dawn heleos 2 mals detector, by Wyatt Technology (1 mentions)
Eclipse3 system, by Wyatt Technology (1 mentions)
20 protocols using optilab t rex differential refractive index detector
1
Protein Characterization by MALS
2
SEC-MALLS Analysis of Membrane Protein Complexes
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SMA:AcrB and A8–35 exchanged (A8–35_Ex) samples were dialysed into 300 mM NaCl, 50 mM Tris-HCl, pH 8.0 (SECB) prior to SEC-MALLS experimentation. DDM exchanged (DDM_Ex) samples were dialysed into SECB plus 10% glycerol and 0.025% DDM (DDM-SECB) to decrease the DDM concentration. For the protein-free lipid particles, 5 mg of E. coli total lipid extracts (Avanti Polar Lipids Inc., U.S.A.) were suspended by sonication in SECB, and split into two for the addition of 2.5% SMA and 2.5% A8–35. SEC-MALLS experiments were performed using a Superose 6 5/150 column pre-equilibrated with SECB for SMA:AcrB and A8–35_Ex samples, and DDM-SECB without glycerol for DDM-containing samples. The data were collected on a DAWN 8+ multi-angle light scattering (LS) detector, an Optilab T-rEX differential refractive index (dRI) detector and UV-absorbance (UV) detector (Wyatt Technology), and samples were run at a flow rate of 0.2 ml/min. Astra 6.2 software was implemented for molar mass calculations. The absorbance value at 0.1% OD280nm for full-length AcrB(His)8 was given as 0.79 g/l and a refractive index increment (dn/dc) value of 0.185 ml/g was applied to the protein component - AcrB. A8–35 and DDM dn/dc modifiers were set at 0.15 and 0.143, respectively, for the surfactant components.
3
Molecular Weight Determination of DNAJB6 Variants
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Solutions of DNAJB6 WT, LGMDD1 disease mutants, or DNAJB6 ΔST (100 µM) were separated by size exclusion chromatography on a Superose 6 Increase 10/300 GL column. Molecular weights were determined by multi-angle laser light scattering using an in-line DAWN HELEOS detector and an Optilab T-rEX differential refractive index detector (Wyatt Technology Corporation). Calculation of molecular weights was performed using the ASTRA software package (Wyatt Technology Corporation).
4
Asymmetric Flow Field-Flow Fractionation
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An Eclipse DualTec AF 4 system (Wyatt Technology, Santa Barbara, CA) was coupled inline to a UV/Vis diode array detector (Model 1260, Agilent Technologies, Santa Clara, CA), a HELEOS-II multiangle laser light photometer, and an Optilab T-Rex differential refractive index detector (Wyatt Technology). The AF 4 channel was a vendor-supplied "short" channel with 350 mm thick "wide" (Mylar) spacer; an Ultracel 10 kDa regenerated cellulose (Millipore, Burlington, MA) ultrafiltration membrane served as the accumulation wall. Samples were introduced into the AF 4 separation channel via an Agilent 1260 autosampler (Santa Clara, CA) with a focus position of 12% of the channel length. The focusing was accomplished by flowing 0.2 mL/min of buffer into the channel inlet and 1.3 mL/min of buffer through the channel outlet for 5 min. After the samples were introduced and focused against the ultrafiltration membrane, they were eluted from the column in a size selective manner with a channel flow of 1.0 mL/min while the cross flow was linearly ramped from 3.0 mL/min to 0 mL/min over 45 min. Post separation the channel was rinsed for 5 min with 1.0 mL/min channel flow and 0 mL/min crossflow and the injector "on" to rinse out the sample loop.
5
Lignin Molar Mass Analysis by MALS
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The molar mass
analysis was done by means of multiangle light scattering
(MALS) in accordance with Zinovyev et al.42 (link) In brief, 10 mg of lignin was dissolved in 1 mL of DMSO/LiBr (0.5%
w/v). After complete dissolution, the sample was filtered through
a 0.45 μm PTFE syringe filter. The SEC analysis was performed
on an Ultimate 3000 system, consisting of autosampler column oven
(all Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.), HPLC Pump
Series P580 (Dionex Softron GmbH, Germering, Germany), HELEOS I MALS
detector operating at 785 nm, and an Optilab T-rEX differential refractive
index detector, λ = 633 nm (all Wyatt Technology, Santa Barbara,
U.S.A.) under the following conditions: 35 °C column temperature,
10 μL injection volume, 0.5 mL min–1 flow
rate, and 65 min run time. For the separation, three Agilent PolarGel
M columns (7.5 × 300 mm with 5 μm particle size) and a
precolumn (7.5 × 50 mm) were used. The data was processed with
Astra 7.3.
analysis was done by means of multiangle light scattering
(MALS) in accordance with Zinovyev et al.42 (link) In brief, 10 mg of lignin was dissolved in 1 mL of DMSO/LiBr (0.5%
w/v). After complete dissolution, the sample was filtered through
a 0.45 μm PTFE syringe filter. The SEC analysis was performed
on an Ultimate 3000 system, consisting of autosampler column oven
(all Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.), HPLC Pump
Series P580 (Dionex Softron GmbH, Germering, Germany), HELEOS I MALS
detector operating at 785 nm, and an Optilab T-rEX differential refractive
index detector, λ = 633 nm (all Wyatt Technology, Santa Barbara,
U.S.A.) under the following conditions: 35 °C column temperature,
10 μL injection volume, 0.5 mL min–1 flow
rate, and 65 min run time. For the separation, three Agilent PolarGel
M columns (7.5 × 300 mm with 5 μm particle size) and a
precolumn (7.5 × 50 mm) were used. The data was processed with
Astra 7.3.
6
Analytical Scale AF4 Separation
7
Characterizing Protein Complex Assemblies
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Purified MTA1:RBBP4, HDAC1:MTA1 and MTA1:HDAC1:RBBP4 complexes that had been gel filtrated were concentrated to 1 mg/ml and reapplied to the Superdex S200 column (GE Healthcare, UK). The mass of each protein complex was calculated immediately on elution with an Optilab T-rEX differential Refractive Index detector coupled to a DAWN HELEOS MALS detector (Wyatt Technology).
8
SEC-MALS analysis of polymers
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SEC analysis was performed by using an Ultimate 3000 autosampler, column oven, UV detector (all Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with a Dionex HPLC Pump Series P580 (Dionex Softron GmbH, Germering, Germany), Dawn HELEOS I MALS detectors with lasers operating at either 658 or 785 nm, and an Optilab T‐rEX differential refractive index detector, λ=633 nm (all Wyatt Technology, Santa Barbara, CA, USA). Both MALS detectors were equipped with 18 photodiodes at different measuring angles, with narrow band pass filters (±10 nm for the respective wavelength used, installed on every second photodiode). The separation was performed with an Agilent PolarGel M guard column (7.5×50 mm) and three PolarGel M columns 7.5×300 mm (5 μm particle size). The columns were kept at 35 °C. The SEC system was operated under the following conditions: 0.5 mL min−1 flow rate; 10 μL injection volume; 65 min run time. Data evaluation used ASTRA software, version 6.1.
9
Size-Exclusion Chromatography of Protein-DNA Complexes
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We injected 100 μl samples of Gcf1p, Gcf1p/DNA20, Gcf1p/DNA50 or Gcf1p_CC/DNA50 (Supplementary Table S3 ) at a protein concentration of 2 mg/ml onto a Superdex 200 10/300 column (Cytiva). Complexes were prepared at protein:DNA ratios of 1:1.2 (Gcf1p/DNA20) and 2:1.2 (Gcf1p/DNA50 and Gcf1p_CC/DNA50). Column equilibration and running buffer for isolated Gcf1p was 750 mM NaCl, 50 mM Tris–HCl pH 8.0, and 20 mM NaCl, 50 mM Tris–HCl pH 8.0 for the complexes. For both cases the flow rate was 0.5 ml/min at RT. The column was coupled to a MALLS system including a DAWN-HELEOS-II detector (Wyatt Technology) with a laser emission wavelength of 664.3 nm. Peak concentrations were measured with an Optilab T-rEX differential refractive index detector (Wyatt Technology) assuming a dn/dC of 0.185 ml/g. Molecular weights (MWs) and complex ratios were determined using conjugate calculations in ASTRA 6.0.5.3 (Wyatt Technology). We also measured absorbance at 280nm. Experiments were carried out at the Automated Crystallography Platform at the Barcelona Science Park.
10
Synthesis and Characterization of Cationic Polymers
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HPMA (103.6
mg, 0.72 mmol), MA-SS-AMD (6 ) (347 mg, 0.36 mmol), AIBN
(11.3 mg), and DMF (2 mL) were added into a 5 mL ampule. After bubbling
with nitrogen for 30 min, the ampule was sealed and the mixture was
heated at 50 °C for 24 h. The polymer was precipitated into cold
diethyl ether, centrifuged, and dried in air for 4 h. The dried polymers
were mixed with trifluoroacetic acid (10 mL) and stirred at room temperature
for 3 h. After trifluoroacetic acid was removed under reduced pressure,
the remaining polymer solution was precipitated by dropwise addition
into cold diethyl ether. The polymer was isolated by filtration, dried
in vacuum, and then dissolved in water and dialyzed against 1 M HCl
followed by final lyophilization. The molecular weights and molecular
weight distribution of the polymers were determined by size-exclusion
chromatography (TSK-GEL PWXL column, Toshoh Bioscience)
equipped with a miniDAWN TREOS multi-angle static light scattering
detector (Wyatt) and Optilab T-rEX differential refractive index detector
(Wyatt) using 0.5 M sodium acetate buffer (pH 5) as the mobile phase.
mg, 0.72 mmol), MA-SS-AMD (
(11.3 mg), and DMF (2 mL) were added into a 5 mL ampule. After bubbling
with nitrogen for 30 min, the ampule was sealed and the mixture was
heated at 50 °C for 24 h. The polymer was precipitated into cold
diethyl ether, centrifuged, and dried in air for 4 h. The dried polymers
were mixed with trifluoroacetic acid (10 mL) and stirred at room temperature
for 3 h. After trifluoroacetic acid was removed under reduced pressure,
the remaining polymer solution was precipitated by dropwise addition
into cold diethyl ether. The polymer was isolated by filtration, dried
in vacuum, and then dissolved in water and dialyzed against 1 M HCl
followed by final lyophilization. The molecular weights and molecular
weight distribution of the polymers were determined by size-exclusion
chromatography (TSK-GEL PWXL column, Toshoh Bioscience)
equipped with a miniDAWN TREOS multi-angle static light scattering
detector (Wyatt) and Optilab T-rEX differential refractive index detector
(Wyatt) using 0.5 M sodium acetate buffer (pH 5) as the mobile phase.
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