Nativepage 4 16 bis tris protein gel

Purified thylakoid membranes were used for two-dimensional Phos-tag SDS-PAGE. Purified thylakoid membranes were resuspended in buffer [25 mM Bis–Tris, 20% (w/v) glycerol, pH 7.5] to a concentration of 2 mg/ml chlorophyll. To solubilize thylakoid membranes, an equal volume of n-dodecyl ß-D-maltoside was added to a final concentration of 1% (w/v). After centrifugation at 14,000 × g for 5 min, NativePAGE™ 5% G-250 Sample Additive (Thermo Fisher Scientific Inc.) was added to the supernatant according to the manufacturer’s instructions, and samples were loaded onto a NativePAGE™ 4–16% Bis–Tris Protein gel (Thermo Fisher Scientific Inc.) Electrophoresis was performed at 4°C overnight at 50 V. The gel lane was then excised from the gel and incubated in equilibration buffer [50 mM Tris–HCl, 6 M urea, 2% (w/v) SDS, 0.05% (w/v) BPB, 10 mM dithiothreitol] for 30 min at 37°C. Then proteins were separated using Phos-tag SDS-PAGE or conventional SDS-PAGE.

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).