Punk dls unit

CSANs were formed by adding a 1.1-3.0 fold molar excess of the desired chemical dimerizer – either bisMTX or aizde-bisMTX – to a solution of targeted DHFR² fusion protein monomers in PBS. Consistent with our previous studies, CSAN oligomerization occurs within minutes of adding the dimerizer.^{27 (link)} Cryo-EM samples were prepared using a Vitrobot Mark IV (FEI). Briefly, 3 μL of CSANs in PBS was applied to a lacey formvar/carbon grid (Ted Pella, Inc.; Cat: 01883) in a humidified chamber, blotted, and plunged into liquid ethane for vitrification. Grids were imaged on a Tecnai Spirit G2 BioTWIN (FEI) equipped with an Eagle 2k CCD camera (FEI) under a high tension of 120 kV. Images were analyzed in ImageJ and, for the size distribution analysis, only nanoparticles with ≥70% circularity were included. For DLS, 60 μL of CSANs in PBS was loaded into a cuvette and analyzed on a Punk DLS unit (Unchained Labs). Hydrodynamic diameter values represent the mean ± standard deviation of at least three measurements.

CSANs were formed by adding a 3-fold molar excess of the desired chemical dimerizer (either bisMTX or DOTA-bisMTX) to a solution of DHFR²-Fn3 fusion protein monomers in PBS. Consistent with our previous studies, CSAN oligomerization occured within 30 minutes of adding the dimerizer.^{41 (link)} Oligomerized CSANs were analyzed by SEC (as described above) and dynamic light scattering (DLS). For DLS, 60 µL of CSANs in PBS was loaded into a cuvette and analyzed on a Punk DLS unit (Unchained Labs). Hydrodynamic diameter values represent the mean ± standard deviation of at least three measurements. SEC and DLS results were consistent with our previous analyses of Fn3-targeted CSANs, including cryo-electron microscopy (cryo-EM) evidence of nanoring formation.^{43 (link), 44 (link)}