Wyatt dawn heleos 2

Size exclusion chromatography with multiangle light scattering detection (SEC‐MALS) experiments were carried out at 25 °C with multiangle light scattering detector (MALS; Wyatt Dawn Heleos‐II) and a size exclusion chromatography (Superdex 200 Increase 10/300 GL column) for measuring the relative molecular mass of protein. Protein samples (˜2 mg·mL⁻¹, 100 μL) were injected in a buffer containing 25 mm Tris–HCl (pH 8.0) and 150 mm NaCl. ASTRA software (Wyatt Technology, Santa Barbara, CA, USA) was used to calculate the molecular weight of protein.

GPC with a multi-angle laser light scattering detector (Wyatt Dawn HELEOS II, Wyatt Technology, Santa Barbara, CA, USA) plus a differential refractometer detector (Waters Model 2414, Waters Corp., Milford, MA, USA) was utilized to determine the molecular weight and polydispersity of the copolymers (in THF solvent). ¹H NMR spectra were recorded on a Varian MERCURY plus-400 (400 MHz) spectrometer (Agilent, Santa Clara, CA, USA) using CDCl₃ solutions, with chemical shifts reported in ppm relative to the residual deuterated solvent.

SEC–MALS experiments were performed using the Wyatt Dawn Heleos II multiangle light-scattering detector (Wyatt Technology) coupled to an AKTA Purifier UPC10 FPLC protein purification system and the Superdex 200 size-exclusion column (GE Healthcare). Protein molecular masses of the individual peaks observed in the size-exclusion chromatograms were analysed by static light scattering in conjunction with their corresponding refractive indices, using an online refractometer connected downstream of the static light scattering detector (Wyatt Optilab rEX). The proteins were prepared at 5 mg ml⁻¹ in a buffer (20 mM HEPES pH 7.5, 150 mM NaCl). HMBPP was prepared in 0.5 mM. The experiments were performed with a running buffer (20 mM HEPES pH 7.5, 150 mM NaCl) at a flow rate of 0.5 ml min⁻¹. A standard value of the refractive index, dn/dc = 0.185 ml g⁻¹, was used for all proteins.

Except where otherwise noted, a Superdex S75 10/30 analytical column (GE Healthcare) was equilibrated with 20 mM MES pH 6.5, 200 mM NaCl, 1 mM dithiothreitol at a flow rate of 0.5 ml/minute. A total of 100 μl of protein sample (at 2–5 mg/ml) was injected, and refractive index and light scattering profiles were recorded inline using Wyatt rEX Optilab and Wyatt Dawn HELEOS-II instruments. Data were analysed using ASTRA software version 5.3.4.14 (Wyatt Instruments), using a Zimm model. Light scattering detectors were normalized on a sample of BSA, and dn/dc chosen in the range 0.164–0.180 to give the correct mass for BSA. All SEC MALLS experiments of TRBP and PACT constructs were conducted at least twice.

Proteins were separated on a sodium dodecyl sulfate polyacrylamide 8% gel (SDS-PAGE). Total protein concentration of each supernatant sample was determined using the Bradford method and 20 μg of protein was loaded into each well. An 8% SDS-PAGE gel was run at a constant 10 watts in a PowerPacTM HC (BIO-RAD) using SDS–PAGE running buffer (25 mM Tris base, 200 mM glycine, 0.1% w/v SDS). The gel was stained with Coomassie G250 to visualize the protein bands.
Purified Netrin-1 was analyzed for particle size distribution and hydrodynamic radius by DLS a Zetasizer Nano S (Malvern Instruments). The protein solution was dialyzed into 50 mM Tris, pH 7.5, and 200 mM NaCl and concentrated to varying concentrations ranging from 0.5 to 9.0 mg/mL for measurements. Measurements were done in triplicates at 20°C.
Furthermore, SEC-MALS measurements of Netrin-1 were performed. For this, protein was concentrated to 5 mg/mL in 50 mM Tris, pH 7.5, and 200 mM NaCl and 300 μL samples were run on a Superose 6 Increase 10/300 (Cytiva) column at 0.3 mL/min. MALS measurements were taken using a Wyatt Dawn Heleos II (Wyatt Technology). For calibration and analysis, bovine serum albumin (BSA) was concentrated to 6 mg/mL and also measured on the same system.

The weight-average molecular weight (Mw), number-average molecular weight (Mn = ∑NM/∑N, N: molecular weight of each component; M: the number of moles of each component), polydispersity (Mw/Mn) and chain conformation of LBPs were estimated by high-performance size exclusion chromatography using an instrument equipped with a multi-angle laser light-scattering system with a refractive index detector (HPSEC-MALLS-RI, Wyatt Dawn Heleos-II, Wyatt Technology, Santa Barbara, CA, USA) according to the method described by Arakawa [50 ], with some modifications. Isocratic elution with 0.20M NaCl solution at a flow rate of 0.5 mL/min was performed on a Shodex SB-804 HQ and SB-806 HQ (Showa Denko KK, Tokyo, Japan) at 25 °C. Sodium chloride solution (0.20M NaCl) was used as the mobile phase at a flow rate of 0.5 mL/min. The injection volume was 50 µL and running time was 100 min. ASTRA software (version 7.1.2, Wyatt Technology) was used to obtain the data.

To further detect the molecular weight of the protein samples, MALLS was applied as it measures the light scattered by a sample into a plurality of angles. In our study, the purity and molecular weight of purified EBNA1‐HBc VLP were evaluated by HPSEC‐MALLS. HPSEC analysis was performed in Shimadzu UHPLC XR system (Shimadzu, Japan), equipped with TSKgel G4000SWXL column (TOSOH Bioscience, Japan). Fifty microliters of sample was loaded and eluted at 0.8 ml/min with 50 mM phosphate buffer (PB) with 100 mM sodium sulfate, pH 7.4. Elution buffer was degassed and filtered by 0.22 μm Millipore membrane (Pall Corporation, USA). The retention time and absorbance at 280 nm were recorded. The HPSEC system was coupled to a MALLS Wyatt DAWN HELEOS II and Optilab T‐rEx (Wyatt Technology, USA) for the detection of the molecular weight. The data were processed with ASTRA software (v. 6.1).