Prior to measurement, glass slides (24 × 50 mm #1.5 special, Menzel-Gläser) were reacted with 3-aminopropyl-triethoxysilane (APTES) to produce an amino-silane functionalized surface. Slides were plasma cleaned under O2, then dipped in a 5% APTES solution in acetone. Excess APTES was removed by rinsing in acetone. The slides were then baked at 110 °C before washing with isopropanol. 5 μM of either apo-NET1ΔC or NET1ΔC with equimolar HO-dp8 or HO-dp10 were incubated overnight in 50 mM tris pH 8.5, 200 mM NaCl at 4 °C. 0.2 μl of NET1ΔC-dp8 or NET1ΔC-dp10 were diluted in 9.8 μl of buffer directly on APTES functionalized glass slides in a 3 mm diameter 1 mm deep culture well gasket (Grace Bio-Labs), for a final concentration of 100 nM. Interferometric videos were taken on a prototype mass photometry system and processed as described32 (link). Briefly, a 477 nm laser focussed to 1.5 μm was passed through a beam splitter and swept across the sample to generate an interferometric video of the buffer-glass interface. Frames were collected at 1000 Hz, and a differential video generated by subtracting each frame from the previous. In these videos, the interaction of the protein with the glass is clearly visible as a spot, where the intensity of the spot is directly proportional to the protein mass. Pixels were pre-binned 3 × 3 and frames were fivefold time averaged during acquisition, giving a final pixel size of 70.2 nm and a frequency of 200 Hz. During processing, videos were subjected to a further fivefold frame averaging. Masses were calculated via a calibration curve using alcohol dehydrogenase, β-amylase, and Protein A. For NET1ΔC w/o GAG, a total count of 8072 were measured with a monomer population of 92.99%. For the NETΔC-dp8 complex, 7192 counts revealed a monomer-dimer ratio of 86.37% to 12.39%. The NET1ΔC-dp10 complex was studied with 17075 total counts revealing a monomer-hexamer ratio of 79.45% to 3.38%.
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