Coomassie brilliant blue g250

DMPC and POPC were kind gifts from Lipoid (Ludwigshafen, Germany). SMA(2:1) (hydrolysed from styrene/maleic anhydride (2:1), tradename Xiran SZ30010) and SMA(3:1) (Xiran SL25010 S25) copolymer solutions were kind gifts from Polyscope (Geleen, Netherlands). DIBMA (Sokalan CP 9) was kindly provided by BASF (Ludwigshafen, Germany). D₂O was purchased from Deutero (Kastellaun, Germany) and NaCl from VWR (Darmstadt, Germany). 85% (w/v) H₃PO₄ in D₂O and Na₂HPO₄ were from Sigma–Aldrich (Steinheim, Germany), and Coomassie Brilliant Blue G250, NaH₂PO₄, ethylenediamine tetraacetic acid (EDTA), sodium dodecyl sulphate (SDS), tris(hydroxymethyl)aminomethane (Tris), and Tris–HCl were from Carl Roth (Karlsruhe, Germany). All chemicals were purchased in the highest purity available.

ADP, aldolase, amyloglucosidase, ATP, bovine serum albumin, di-sodium hydrogen phosphate (Na₂HPO₄), fructose-6-phosphate, glycerol-3-phosphate dehydrogenase, glucose, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, imidazole, magnesium chloride hexahydrate (MgCl₂×6H₂O), phosphoenolpyruvate, potassium chloride (КСl), potassium dihydrogen phosphate (KH₂PO₄), sodium azide (NaN₃), sodium chloride (NaCl), sodium pyruvate, triose-phosphate isomerase and were purchased from Sigma-Aldrich (USA). NAD⁺, NADH, NADP⁺, NADPH, EDTA, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol, sodium fluoride (NaF), lactate dehydrogenase, Coomassie Brilliant blue G-250 and Triton X-100 were purchased from Carl Roth (Karlsruhe, Germany). Diagnostic kits for determination of glucose and triacylglycerides were from Private Joint-Stock Company «Reagent» (Dnipro, Ukraine). Alpha-ketoglutaric acid was from Protista AB (Sweden).

After harvest, patch 2 (Fig. 1A) was transferred to sterile 10 mL glass tubes containing 2 mL of 1x PBS buffer pH 7.4, the tubes vortexed for 1 min and then sonicated (3 times for 30 s with 1 min pause each time) in a sonication bath (SONOREX SUPER RK 255 H, BANDELIN, Germany) at 35 kHz ultrasound wave. The cell suspension was then transferred into 2 mL tubes and centrifuged at 16,000 × g and 4 °C (Eppendorf, Germany) to pellet the cells. The cells were lysed in 50 µL lysis buffer (10 mM Tris; 5 mM EDTA; 0.29% NaCl; and 0.4% SDS) and processed as previously described [40 (link)]. A Bradford assay was used to estimate protein concentrations. For the SDS-PAGE, 75 µg of protein was precipitated with an equal volume of 20% trichloroacetic acid (TCA, Sigma) for 16 h at 4 °C. The proteins were then separated in a 12% acrylamide gel using the Hoefer^TM mini–Vertical Electrophoresis system (Thermo Fisher Scientific, USA) and the Laemmli-buffer system [40 (link)]. The protein bands were visualized by staining the gel in colloidal Coomassie Brilliant blue G-250 (Roth, Kassel, Germany). Protein bands were excised from the gel and subjected to in-gel tryptic digestion as described previously [40 (link)]. Tryptic peptides of all gel slices were then extracted for LC-MS/MS analysis.