Dynamics v6

Dynamic light-scattering experiments were performed on a DynaPro Titan instrument (Wyatt Technologies) at 20 °C. CgGDE was measured at a concentration of 20 mg ml⁻¹, in a buffer containing 20 mM Tris pH 7.5, 200 mM NaCl and 2 mM DTT. Data were analysed with the DYNAMICS V6 software (Wyatt Technologies).

Dynamic light scattering experiments were performed on a DynaPro NanoStar instrument (Wyatt Technologies) at 25 °C. KlRad5 was characterized at a concentration of 1 mg/ml, in a buffer containing 20 mM Tris (pH 7.5), 200 mM sodium chloride and 2 mM DTT. Data were analyzed with the DYNAMICS V6 software (Wyatt Technologies).

DLS was used to estimate the size of IgG conformers present in unfractionated, SEC- and affinity column-fractionated IVIg, pAb and mAb samples. DLS was carried out at room temperature using a DynaPro plate reader (Wyatt Technologies Corp., Santa Barbara, CA) [38 (link)]. Briefly, 100 μl aliquots of ~0.02 to ~1 mg/ml antibody samples and buffer blanks were added to wells of a polystyrene flat bottom 96 well half-area black microplate (cat# 3881, Corning, Tewksbury MA). DLS readings were performed using 6 to 20 10s acquisitions with auto-attenuation. The hydrodynamic radius (R_h), polydisperse indice, and apparent molecular weights for IgG conformers in each sample were calculated using DYNAMICS V6 software (version 7.1.7.16, Wyatt, Santa Barbara, California).

DLS measurements were taken using a DynaPro Plate reader (Wyatt Technology) equipped with a temperature control unit. Purified BSA (Sigma Aldrich), hTPI⁺, and hTPI^M80T were diluted to concentrations of 3.75 μM, 15 μM and 30 μM in 100 mM triethanolamine (TEA); pH 7.6. Three 75 μl aliquots were loaded onto a 384-well microplate and read at 37°C. Ten measurements were taken per sample and Dynamics V6 software (Wyatt Technology) was used to process the scattering data, generating autocorrelation functions. Autocorrelation functions were then analyzed to obtain the hydrodynamic radii. Student’s T test was used to compare samples. DLS experiments were performed three times.

The apparent hydrodynamic radii of the proteins were measured using dynamic light scattering (DLS) in a DynaPro MS-X DLS instrument (Wyatt, Santa Barbara, CA, USA). Dynamics v6 software (Wyatt Technology Corporation, Santa Barbara, CA, USA) was used in data collection and processing. Sets of DLS data were measured at 25 °C with an average number of 50 acquisitions and an acquisition time of 10 s. Measurements were carried out in 50 mM sodium phosphate buffer pH 7.4 and 50 mM glycine/HCl buffer pH 2.5.
Static scattering intensities were measured in a Malvern μV instrument (Malvern Panalytical, Malvern, UK) at 25 °C, in 50 mM sodium phosphate buffer pH 7.4, at different concentrations of protein in a range of 0.2 to 3.5 mg mL⁻¹. The intensities were analyzed using the Debye plot as represented by Equation (1),

valid for particles significantly smaller than the wavelength of the incident radiation, where the K is an optical constant of the instrument, c is the particle mass concentration, R₉₀ is the Rayleigh ratio of scattered to incident light intensity, M_w is the weight-averaged molar mass, and A₂ is the 2nd virial coefficient that is representative of inter-particle interaction strength. M_w can be determined from the intercept of the plot.

For size exclusion chromatography, 0.1 mL of purified A2-G protein (0.12 mg/mL in 0.137 M NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.2) was loaded onto a BioSeb SEC-S 3000 column (300 × 7.80 mm; Phenomenex, Aschaffenburg, Germany) at 4 °C, equilibrated with protein storage buffer (0.137 M NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.2). The flow rate was 0.5 mL min⁻¹ and chromatograms were obtained at 225 nm (UV-VIS) wavelength. The molecular mass of defined peaks of A2-G were calculated based on the elution profile of a gel filtration protein standard (66 kDa: Bovine serum albumin (1 mg); 29 kDa: Carbonic Anhydrase (1 mg); 12.4 kDa: Cytochrome C (1 mg); Sigma Aldrich, Taufkirchen, Germany) performed under identical buffer conditions.
Dynamic light scattering (DLS) was performed on a DynaPro-NanoStar device using 20 µL of 0.1 mg/mL A2-G protein in a disposable micro cuvette (Wyatt Technology; Hollister Avenue, Santa Barbara, CA, USA) at 20 °C in 0.137 M NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7. Twenty acquisitions, with a five-second acquisition time for each measurement, were carried out with a laser power of 50%. Hydrodynamic radius was calculated using dynamics V6 software (Wyatt Technology Corporation, Santa Barbara, CA, USA).

Fusion of isolated synaptic vesicles with ΔN-SNARE-proteoliposomes was followed by dynamic light scattering using a DynaPro® instrument (Wyatt Technology). First, the size distribution of freshly isolated synaptic vesicles and ΔN-SNARE-proteoliposomes were recorded separately. Membrane fusion was then initiated by mixing 25 μl of synaptic vesicles with 25 μL of 5-times diluted ΔN-SNARE-proteoliposomes in a black quartz cuvette placed in the DynaPro® instrument. The mixture was incubated at 37 °C and size distributions were recorded at 15, 60 and 90 min. Ten sequential measurements of 5 s were performed at each time-point using a scattering angle of 90°. The data were processed using the Dynamics V6 software (Wyatt Technology). The radii and the size distributions were calculated with the regularisation algorithm as specified in the software.