Optilab dsp

FL and Δ76 1CGrx1 were analyzed by SEC-MALS as previously described^{56 (link)}. The samples were loaded onto a Superdex 75 10/300 GL SEC column (GE-Healthcare) equilibrated with 20 mM Tris pH 8.0, 100 mM NaCl and 5 mM DTT connected to a multiple-angle laser light (690.0 nm) scattering DAWN EOS photometer (Wyatt Technology) and a refractive index detector (Optilab DSP, Wyatt). The specific refractive index increment (dn/dc) for the protein was taken as 0.185 mL/g^{57 (link)}. The value of 1.331 for the solvent refractive index and the concentration of the eluted protein were determined using the refractive index detector. The weight average molecular masses, M_w, were determined across the entire elution profile in the intervals of 0.2 s from MALS measurement using the ASTRA software (Wyatt Technology). A Rayleigh–Debye–Gans light scattering model was used to determine M_w, using a Zimm plot. The uncertainties on M_w are a measure of the statistical consistency of the MALS data, obtained combining the standard deviations calculated for each slice in the analyzed peaks. Data analysis was performed using Astra version 5.3.4 following the manufacturer’s instructions.

The molar mass of the highly oligomerized CIDE domain of DREP2 was determined by MALS. The target protein was injected onto a Superdex 200 HR 10/30 gel filtration column (GE Healthcare). The chromatography system was coupled to a three-angle light scattering detector (mini-DAWN EOS) and refractive index detector (Optilab DSP) (Wyatt Technology, Santa Barbara, CA, USA). Data were collected every 0.5 s at a flow rate of 0.2 mL/min and analysed using the ASTRA program, which gave the molar mass and mass distribution (polydispersity) of the sample.

Analytical-scale HPLC was performed with an Agilent 1200 series system on octadecyl carbon chain (C18)–bonded silica columns (5-μm pore size, 4.6 × 250 mm, 218TP54, Vydac). The solvent system was HPLC-grade H₂O (W/0106/PB17, Fisher Scientific) and HPLC-grade acetonitrile (ACN; A/0627/PB17, Fisher Scientific). Buffer A consisted of HPLC-grade H₂O containing 0.1% (v/v) HPLC-grade TFA (T/3258/04, Fisher Scientific), and buffer B consisted of 80% (v/v) ACN in HPLC-grade H₂O containing 0.1% (v/v) TFA.
Mass spectrometry analysis was performed on the Agilent Series 1100 LC-MS system in positive mode of ESI. The solvent system was as follows. Buffer A consisted of LC-MS grade H₂O (W/0112/17, Fisher Scientific) containing 0.1% (v/v) formic acid (06440, Fluka), and buffer B consisted of 80% (v/v) ACN in MS-LC–grade H₂O containing 0.1% (v/v) formic acid. Data analysis was performed with LC/MSD ChemStation software.
SEC-MALS was performed on a Superdex 200 5/150 GL column (GE Healthcare) in SEC-MALS buffer (50 mmol/liter Tris-HCl (pH 7.4), 5 mm MgCl₂, 1 mmol/liter CaCl₂, 100 mm NaCl, and 100 mmol/liter tris(2-carboxyethyl)phosphine). Recombinant troponins were extensively dialyzed against SEC-MALS buffer before experiments. Light scattering and refractive index were measured on a Mini DAWN and OPTILAB DSP (Wyatt Technology, UK), respectively. Data were analyzed with custom-written software.

¹H NMR (nuclear magnetic resonance) spectra of all the samples were characterized on a Mercury VX 300 M spectrometer in DMSO-d₆ at 25°C using tetramethylsilane (TMS) as an internal reference. Size exclusion chromatography and multi-angle laser light scatting analysis (SEC–MALLS) were used to determine the molecular weight and molecular weight distribution. A dual detector system consisting of a MALLS device (DAWNEOS, Wyatt Technology) and an interferometric refractometer (a differential refractive index detector, Optilab DSP, Wyatt Technology) was used. 0.1 M NaNO₃ served as the eluent at a flow rate of 0.3 ml/min. The column temperature was fixed at 25°C and the MALLS detector was operated at a laser wavelength of 690 nm.
NP morphology was investigated on the transmission electron microscopy (TEM) machine (JEM-2100 microscope) at an acceleration voltage of 100 keV. The samples were prepared by placing a droplet of micelles solution on a copper grid with formvar film, which was stained by a 0.2% (w/v) phosphotungstic acid solution, and then slowly dried in air before visualization.
The hydrodynamic particle size and size distribution of the micelles were measured by dynamic light scattering (DLS) at 25^oC using Nano-ZS ZEN3600 (Malvern instruments). Data were shown as mean ± standard deviation based on three independent measurements.

The molecular features of LP4 were confirmed with high-performance size exclusion chromatography (Waters 2695, Milford, MA, United States) coupled with a multi-angle laser light scattering detector (DAWN HELEOS-II, Wyatt Technology, Santa Barbara, CA, United States) and refractive index detector (Optilab DSP, Wyatt Technology, Santa Barbara, CA, United States) (SEC–MALLS–RID). LP4 was analyzed on a Ultrahydrogel 2000 (Waters, Milford, MA, United States) column and eluted with 0.1 mol/L NaNO₃ (0.5 ml/min). The column temperature was kept at 35°C. The data were analyzed with ASTRA 5.3.4.20 software.