Nativemark protein ladder

All samples were measured in 1X HBS-N buffer (10 mM HEPES pH 7.4, 150 mM NaCl) using the Refeyn OneMP mass photometer (Refeyn Ltd.) with a 60 s acquisition time. The resulting histograms were fitted to Gaussian distributions using DiscoverMP (Refeyn Ltd.) to extract peak contrast and relative amount of each peak (n = 3). Contrast-to-mass conversion was achieved by calibration using NativeMark protein ladder (Thermo Fisher Scientific). Three protein species (with specified masses) were fitted to corresponding Gaussian distributions to extract a linear relation between mass and contrast.

Proteins from parasites were solubilised in Native PAGE Sample Buffer (Thermo Fisher Scientific) containing 2 mM EDTA, 1 × cOmplete protease inhibitors (Sigma) and 1% (v/v) TX-100 (Sigma) at 2.5 × 10⁵ parasites per μL. Samples were separated by Clear Native PAGE on a NativePAGE 4–16% Bis-Tris protein gel (Thermo Fisher Scientific) using BN-PAGE buffers prepared without cathode buffer additive, but with sodium deoxycholate (0.05% w/v, Sigma, D6750) and n-dodecyl β-D-maltoside (0.01% w/v, Sigma, D4641). After separation, the lane containing the NativeMark protein ladder (Thermo Fisher Scientific) was removed and stained with Coomassie. An in-gel Complex IV activity assay was performed on the remaining lanes as described in [27 (link)]. Briefly, the gel was equilibrated in 50 mM KH₂PO₄ buffer (pH 7.2) for 15 minutes before incubating in freshly made Complex IV activity buffer (KH₂PO₄ (50 mM, pH 7.2), cytochrome c (0.1% w/v from horse heart, Sigma, C2506), catalase (0.1% w/v from bovine liver, Sigma, C1345), sucrose (7.5% w/v, Sigma, S9378) and 3,3′-diaminobenzidine tetrahydrochloride (DAB, 0.1% w/v, Sigma, D8001) until staining of the no ATc sample was visible. The gel was then fixed in methanol (50% v/v) and acetic acid (10% v/v), re-connected with the Coomassie-stained protein ladder and imaged using a ChemiDoc MP imaging system (BioRad).