Acquiremp v 2

Mass photometry experiments were carried out using a Refeyn One^MP instrument (Oxford, UK). Microscope coverslips were cleaned several times with H₂O and isopropanol and then air-dried. A silicone gasket was placed onto the clean coverslip and 19 µL of buffer B were applied. The focal position was identified automatically using the Refeyn Acquire^MP v.2.4.0 software. 1 µL of purified EXT1-EXT2 at a concentration of 600 nM were added to the buffer drop, resulting in a final protein concentration of 30 nM. Movies with a duration of 60 s, corresponding to 6000 frames, were recorded using a regular field-of-view acquisition setting. Corresponding mass for automatically detected particles was calculated using the Refeyn Discover^MP v.2.5.0 software. Mass kernel density were estimated with a 4.6 kDa bandwidth. Contrast-to-mass calibration was performed using a NativeMark^TM unstained protein standard (Thermo Fisher Scientific), containing proteins with a molecular weight of 66, 146, 480, and 1048 kDa.

Mass photometry analyses were conducted using the Refeyn OneMP device (Oxford, UK). Purified proteins were diluted in the following buffers: XylT1 and SUMO-β3GalT6 in Tris buffer A with 5% glycerol (v/v), β4GalT7 in S200 buffer, and GlcAT-1 in HEPES buffer. Cover slides (24×50 mm, 170 μm) were thoroughly rinsed with demineralized water and isopropanol, and subsequently dried under a clean stream of compressed air. A reusable culture well gasket (Grace Bio-Labs) was placed onto a cover slide. To establish focus, 19 μL of buffer was added to the well and autofocus mode was engaged using the Refeyn AcquireMP v.2.4.0 software. Movies, consisting of 60 s (6,000 frames), were recorded with a standard field-of-view, and the data was processed using DiscoverMP v2.5.0 software. Calibration was performed by adding 1 μL of 1:20 diluted NativeMark Protein Standard (Thermo Fisher Scientific) in buffer. For each acquisition, either 18 μL or 19 μL of buffer were employed to set the focus, followed by the addition of 1 μL to 2 μL of the protein sample to achieve a final concentration between 50 nM and 125 nM. Duplicates were carried out for each sample.