Tris hcl

Phenylmethylsulfonyl fluoride (PMSF), KH₂PO₄, NaCl, ethylenediamine tetraacetic acid (EDTA), ATP, ADP, MgCl_2, β-hydroxybutyrate, phosphoenolpyruvate, sodium L-lactate, sodium pyruvate, imidazole, hydrazine, aldolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, fructose 6-phosphate, glucose 6-phosphate, glucose, G6PDH, КСl, mannitol, bovine serum albumin, glutamate, malate, rotenone, succinate, antimycin A, ascorbate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), and NaN₃ were purchased from Sigma-Aldrich (St. Louis, MO, USA); Tris-HCl, NADP, NADH, NAD⁺, glycine, lactate dehydrogenase were from Carl Roth (Karlsruhe, Germany); diagnostic kits for determination of glucose and triacylglycerides were from Felicit Diagnostics Ltd. (Dnipro, Ukraine). All other reagents were obtained from local suppliers (Ukraine) and were of analytical grade.

Naturally fermented milk samples were directly quenched in 2 mL screw cap tubes in RNA-later (ThermoFisher Scientific) and shipped to TNO Zeist, The Netherlands. Subsequently, 10 μl of sample was transferred to a new 2 mL screw cap tube that contained 300 μl of lysis buffer (Agowa Mag Mini DNA Isolation Kit, LGC Ltd., United Kingdom), 500 μl zirconium beads (0.1 mm; BioSpec products, Bartlesville, Oklahoma, United States), and 500 μl of phenol saturated with Tris–HCl (pH 8.0; Carl Roth GmbH, Germany). Mechanical disruption of bacterial cells was done by bead beating for 2 min in a mini-beadbeater-8 cell disruptor (Merlin Bio-products, Breda, The Netherlands) at setting fast (homogenize). After bead beating, the samples were cooled on ice prior to a 10 min 10,000 rotations per minute centrifugation step. After another phenol extraction step of the aqueous phase, 300 μl of the aqueous phase was transferred to a new centrifugation tube pre-filled with 600 μl binding buffer and 10 μl magnetic beads (Agowa). After mixing, the suspension was left for 30 min to allow binding of the chromosomal DNA to the magnetic beads. After washing the beads, the DNA was eluted from the beads with 65 μl according to the Agowa Mag mini DNA extraction protocol.

We transferred 3 g of homogenized sample material to a 50-mL Falcon tube and added 30 mL extraction buffer (10 mM ammonium penta-borate (Alfa Aesar, Karlsruhe, Germany), 16 mM Tris-HCl (Carl Roth, Karlsruhe, Germany) and 2.5 M urea, pH 8.5) at 20 °C. The sample was mixed and incubated in a water bath at 90 °C for 10 min with brief shaking after every 3 min. After cooling in tap water for 10 min, the samples were centrifuged at 4700× g for 30 min at 4 °C.

1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids, Alabaster, AL, USA), hexadecane (Sigma Aldrich, Darmstadt, Germany), silicone oil AR20 (Sigma Aldrich), α-hemolysin (Sigma Aldrich) and γ-cyclodextrin (Cyclolab, Budapest, Hungary) were used as received. The buffer for protein and γ-cyclodextrin dilution consisted of 1 M KCl (Sigma Aldrich) and 10 mM Tris–HCl (Roth, Karlsruhe, Germany), pH 7.0. The lipid solution was prepared by dissolving DPhPC (200 mg) in chloroform (10 mL, Chempur, Piekary Slaskie, Poland). The chloroform was evaporated under vacuum and the lipid film was re-solubilized in a mixture of 75% (v/v) hexadecane (Alfa Aesar, Karlsruhe, Germany) and 25% (v/v) silicone oil AR20 (Sigma Aldrich) to the final concentration 1 mg/mL.

The six test proteins (BSA, avidin, myoglobin, recombinant protein G, jacalin, and apotransferrin) were characterized and visualized by silver stained SDS-PAGE (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, protein samples were mixed with an equivalent volume of 2x Laemmli buffer, containing 65.8 mM Tris-HCl (pH 6.8, Carl Roth), 26.3% glycerol (v/v, Carl Roth), 2.1% SDS (Carl Roth), 0.02% bromophenol blue (Sigma-Aldrich) and 5.0% 2-mercaptoethanol (Sigma-Aldrich), and heated at 95 ^∘C for 5 min. The protein samples (50 ng in 10 μL) and the color-prestained standard marker (10 ng, broad range, 11–245 kDa, New-England Biolabs, Frankfurt, Germany) were loaded onto the gels (Protean Precast, 4–20%, Bio-Rad, Munich, Germany). The manufacturer’s silver stain kit protocol (Thermo Fisher Scientific) was applied. A ChemiDoc system (Bio-Rad) with Image Lab software 5.2.1 (Bio-Rad) was used for image acquisition. The SDS-PAGE gel image can be found in Figure S6 (Electronic Supplementary Material).

Tissue protein extracts from liver were prepared on ice using the lysis buffer (50 mM Tris-HCl (Carl Roth, Karlsruhe, Germany), 150 mM NaCl (AppliChem, Darmstadt, Germany), 2 mM EDTA (Sigma Aldrich, Munich, Germany), 2 mM NaF (Sigma Aldrich, Munich, Germany), 1 mM Na₃VO₄ (Sigma Aldrich, Munich, Germany), 1% Nonidet P40 BioChemica (AppliChem, Darmstadt, Germany) 1% Triton X-100 (Sigma Aldrich, Munich, Germany) and cOmplete EDTA-free Protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany) and analyzed by western blotting. Equal amounts of protein (50 µg/lane) were loaded. Western blotting was scored by an observer blinded to the groups.