Dextran 40

Yfh1 was expressed in E. coli and purified as described by He et al.42 (link) Purity of the recombinant proteins was checked by SDS–polyacrylamide gel electrophoresis after each step of the purification.
All crowding agents, PEG 20, dextran 40, Ficoll 70 and Ficoll 400 were purchased from Sigma Aldrich. Concentrated solutions to be used as mother solutions to prepare samples at different crowder concentrations were examined for their potential influence on the final ionic strength and pH.

iRFP713 and its variants in the holo- and apoforms were expressed and purified as described previously (Stepanenko et al., 2014 (link)) (Supplemental Methods 1). SDS/PAGE in a 12% polyacrylamide gels was used to confirm that the purity of the target proteins was at least 95% (Laemmli, 1970 (link)). The concentrated protein was stored in 20 mM Tris/HCl buffer, 150 mM NaCl, pH 8.0. The measurements were carried out at a low protein concentration (absorbance was kept less than 0.1, which corresponds to a protein concentration of 0.14 mg/ml) in 20 mM Tris/HCl buffer, pH 8.0, 1 mM tris(2-carboxyethyl)phosphine (TCEP).
GdnHCl, GTC, TCEP, and crowding agents PEG-8000, Dextran-40 and Dextran-70 were purchased from Sigma (St. Louis, MO, USA). The concentration of GdnHCl and GTC in stock solutions was calculated by the refraction coefficient measured by the Abbe refractometer (LOMO, St. Petersburg, Russia).

The m-VROC viscometer
(RheoSense, Inc.) was used to measure the dynamic viscosity of protein
samples, with a water bath to maintain the flow channel at 65 °C.
Stock solutions of Fab, buffer, and excipients were mixed and filtered
through 0.22 μm filters prior to the measurement. The filtered
sample, contained in a Hamilton 0.5 mL syringe, was loaded onto a
syringe jacket. The m-VROC measures the pressure
drop along an array of sensors when a liquid passes through the cell.
The slope (and corresponding R² of the
fit) is calculated from the pressure drop as a function of distance
(essentially shear stress versus shear rate) and is used to calculate
the viscosity. During the measurement, shear rates of up to 18,000
s^–1 were performed with corresponding viscosities
recorded. A water sample was always measured as a reference before
every protein sample to ensure the cleanness of the flow channel.
The reported viscosities were averaged from measurement repeats for
which the “Slope Fit R²”
was >0.99. Chemicals were purchased from Sigma-Aldrich (Dorset,
UK)
with Ficoll 70 (F2878), polyvinylpyrrolidone (PVP40), and dextran
40 (31389) used as crowding agents.
The diffusion coefficient k_d for the bimolecular reaction
rate constant was determined based on eq 6,^{31 (link)} where R is the gas constant and T and μ are the absolute
temperature and viscosity at 65 °C, respectively.