Bca protein quantification assay

Lyophilized supernatant samples (containing secreted proteins) or lyophilized bacterial pellets were resuspended in 4% SDS, 100 mM HEPES, 10 mM DTT and boiled at 95 °C for 10 min with shaking at 2000 rpm to solubilize protein material. 100 μg of protein, as determined by BCA protein quantification assay (Thermo Fisher Scientific), was precipitated by overnight incubation in 80% acetone at −20 °C with shaking. Precipitated proteins were centrifuged at 10,000 G for 10 mins at 0 °C. To ensure removal of SDS, resulting pellets were again suspended and incubated overnight in 80% acetone. Precipitated protein pellets were obtained by centrifugation and dried at 75 °C for 5 mins and stored at −20 °C until analyzed.

Sternohyoid muscles were weighed and homogenised with 8 × 10 s bursts using a general laboratory homogeniser (Omni‐Inc., Kennesaw, GA, USA) in modified ice cold radioimmunoprecipitation assay (RIPA) buffer containing: RIPA (25 mM Tris–HCl pH 7.6, 150 mM sodium chloride, 1% NP‐40, 1% sodium deoxycholate, 0.1% SDS), deionised water, protease inhibitor cocktail (104 mM 4‐benzenesulfonyl fluoride hydrochloride (AEBSF), 80 μM aprotinin, 4 mM bestatin, 1.4 mM E‐64, 2 mM leupeptin, 1.5 mM pepstatin A), phosphatase inhibitor cocktail (200 mM sodium fluoride, 5 mM sodium orthovanadate) and 100 mM phenylmethylsulfonyl fluoride (Sigma Aldrich, Arklow, Wicklow, Ireland) using a 10% w/v ratio. Homogenates were allowed 20 min lyse time on ice with intermittent vortexing at 4‐min intervals. Samples were centrifuged (U‐320R centrifuge Boeckel + Co, Hamburg, Germany) for 20 min at 4°C at 15,366 g and the supernatant was stored −80°C. The protein concentration of each sample was determined using a bicinchoninic acid (BCA) protein quantification assay (Thermo Fisher Scientific), as per the manufacturer's instructions. Absorbance was measured at 562 nm using a SpectraMax‐M3 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

The protein solubility measurement was conducted according to the method of Bera and Mukherjee [27 (link)] with some modifications. To better determine the impact of the pH on the functional properties of CP, the pH of CP dispersion in distilled water (l mg/mL) was increased from 2.0 to 12.0 (intervals of 1.0). The sample solutions were stirred at room temperature for 2 h and then centrifuged at 8500 rpm for 10 min. The content of the protein in the supernatant was measured using a bicinchoninic acid (BCA) protein quantification assay (Thermo Fisher Scientific, Darmstadt, Germany). The protein solubility (%) was calculated by the following equation: $$\mathrm{Solubility} (\%)=\frac{\mathrm{Protein} \mathrm{concentration} \mathrm{in} \mathrm{the} \mathrm{supernatant} \left(\mathrm{mg}/\mathrm{mL}\right)}{\mathrm{Sample} \mathrm{concentration} \left(\mathrm{mg}/\mathrm{mL}\right)}\times 100\%$$

A separate cohort of rats (Sham, n = 12; CIH, n = 12) were euthanised by pentobarbitone (i.v.) overdose and whole brains were removed. The pons and medulla oblongata were separated from the brain, frozen in isopentane at −80 °C and stored at −80 °C until subsequent determination of brainstem cytokine concentrations. Pons and medulla oblongata tissue (Sham, n = 12; CIH, n = 12) were weighed and sonicated (1 ml per 100 mg of tissue) in radioimmunoprecipitation assay (RIPA buffer) (10X RIPA, deionised H₂0, 200Mm sodium fluoride, 100Mm, phenylmethylsulfonylfluoride (PMSF), 1X protease inhibitor cocktail and 1X phosphate inhibitor cocktail). Samples were centrifuged at 10,000 g for 15 min at 4 °C, to pellet membranes and nuclei. The protein concentration of each sample was determined using a bicinchoninic acid (BCA) protein quantification assay (Thermo Fisher Scientific) as per the manufacturer's instructions, at a dilution of 1:10.

The uptake of choline in Huh7.5 cells was determined by using [³H]-choline (Perkin Elmer, Woodbridge, ON, Canada) to evaluate uptake the desired time points. Prior to the addition of radiolabeled choline, Huh7.5 cells seeded in 24-well plates were washed twice with PBS then incubated with Krebs-Ringer-HEPES buffer (KRH; 130 mM NaCl, 1.3 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 10 mM HEPES pH 7.4, 10 mM glucose) for 1 h at 37 °C prior to treatment to remove extracellular choline. Following KRH incubation, [³H]-choline (1 µCi/mL in KRH) was added to the cells for the desired time points (0–30 min) and incubated at 37 °C. For uptake kinetics, cells were incubated in increasing concentrations of non-radiolabeled choline for 10 min. The [³H]-choline was then removed, and the cells were washed twice with KRH buffer, then lysed in 300 µL of 0.1 M NaOH. Cells were flash frozen using liquid nitrogen, thawed then the lysate was scraped and collected. Cell lysate was centrifuged at 20,000× g for 5 min and supernatant was collected. Cellular protein amounts were determined by BCA protein quantification assay according to manufacturer’s instructions (Thermo Fisher Scientific, Ottawa, ON, Canada) and radioactivity was quantified through liquid scintillation counting (LSC). Choline uptake and kinetics were calculated as previously described [28 (link)].