Tris hcl ph 7

Briefly, mitoplasts, prepared from fibroblasts by treatment with 1.2 mg digitonin per mg of protein, and samples containing 100 μg mitochondrial protein were separated on NativePAGE 4%–16% Bis-Tris gels (Thermo Fisher Scientific, Cat# BN1002BOX). For the assessment of complex I activity, the gel was transferred to a solution of 2 mM Tris-HCl pH 7.4 (Sigma-Aldrich, Cat# 10812846001) containing 0.1 mg/mL nicotinamide adenine dinucleotide (Sigma-Aldrich, Cat# 481913-500 MG) and 0.25 mg/mL nitro blue tetrazolium (Sigma-Aldrich, Cat# N5514-10TAB) at 37°C with mild agitation. Banding began to develop within 2 h with an optimal band visualization at >24 h. 10 μg mitochondrial protein were analyzed by Western blot with antibody against TOM20 (as loading control).

Absolute ethanol, allyl isothiocyanate, barium acetate, ß-mercaptoethanol, cetyltrimethylammonium bromide (CTAB), chloroform, ethylenediaminetetraacetic acid (EDTA 500 mM), formic acid (FA), isoamyl alcohol, lead(II) acetate, phenyl isothiocyanate, polyvinylpyrrolidone (PVP), sodium acetate, sodium chloride, and Tris–HCl pH 7.5 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aryl sulfatase from Helix pomatia with 10,000 units (type H-1; EC 3.1.6.1) was obtained from Sigma-Aldrich. DEAE Sephadex A-25 was purchased from Cytiva (Marlborough, MA, USA). Glucotropaeolin potassium salt was obtained from Extrasynthese (Genay Cedex, Rhône, France). Plant Total RNA Mini Kit without DNase was purchased from Geneaid (New Taipei City, Taiwan). High-performance liquid chromatography (HPLC) solvents acetonitrile and methanol were purchased from Daejung (Republic of Korea), and water and dichloromethane were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Tris-HCl pH 7.5, NaCl, CaCl₂, and Triton X-100 were purchased from Sigma-Aldrich (St Louis, MO, USA) for homogenization buffer preparation. EDTA-free proteinase inhibitor cocktail was purchased from Roche Diagnostics GmbH (Mannheim, Germany). A Milliplex MAP multiplex assay panel (human cancer/metastasis biomarker magnetic bead) and human bone RANKL single-plex panel was obtained from Millipore, Merck KGaA (Darmstadt, Germany).

Stable Isotope Labeling by Amino acids in Cell culture minimal medium consisting of 15 mM (NH₄)₂SO₄, 2 mM CaCl₂, 1 μM FeSO₄.7H₂O, 8 mM MgSO₄, 10 μM MnSO₄, 27 mM KCl, 0.6 mM KH₂PO₄, 7 mM C₆H₅Na₃O₇⋅2H₂O (Merck), 50 mM Tris-HCl pH 7.5 (Sigma–Aldrich) supplemented with 0.5% glucose (AppliChem), 0.67 mM glutamic acid (Merck) and 490 μM tryptophan (Sigma–Aldrich), was used to grow a lysine auxotrophic strain of B. subtilis 168 (also referred to as the wild-type or WT). For labeling purposes, the minimal medium was supplemented with 0.025% of the respective isotopically labeled L-lysine – Light, Lys0: ¹²C₆¹⁴N₂ (Sigma–Aldrich); Medium, Lys4: 4,4,5,6-D₄; Heavy, Lys8: ¹³C₆¹⁵N₂ (Euriso-Top). An overnight culture grown until an OD₆₀₀ of 0.5–0.6 was used as a pre-inoculum for the main cultures which were grown at 37°C at 200 rpm and harvested at either the late stationary phase of growth or mid-logarithmic phase of growth. The ΔlysA WT strain was labeled with “Light” lysine, the ΔptkAΔlysA (kinase) strain with “Medium” lysine and the ΔptpZΔlysA (phosphatase) strain with “Heavy” lysine. Cells were harvested by centrifugation at 7000 × g for 10 min. Supplementary Figure 1 depicts the entire (phospho) proteomics workflow employed. A total of four biological replicates were performed for the phosphoproteomics experiments and three biological replicates and the whole proteome analysis.

For ChIRP-MS, the ovarian cell suspension was reversibly cross-linked in situ with formaldehyde (Sangon Biotech, Shanghai, China), followed by cleaving with lysis buffer (50 mM Tris-HCl pH 7.0 (Sigma-Aldrich, St. Louis, MO, USA), 1% SDS (Sangon Biotech), 10 mM EDTA (Sigma-Aldrich), 1 mM PMSF (Sangon Biotech)) and hybridization with biotin-labeled RNA probes (control probe and Gm2044 probe). After the non-specific binding proteins were washed out under strong denaturation conditions, the obtained RNA-binding proteins were identified and quantitatively analyzed by liquid chromatography-tandem MS. Then, the interactive protein profile of lncRNA Gm2044 was constructed and analyzed with gene ontology (GO, http://geneontology.org/), the Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/) and STRING (https://cn.string-db.org/). The probes of lncRNA Gm2044 were listed in Table S3, and the control probe was described in a previous study [17 (link)]. For RNA IP, the corresponding RNA-protein complexes were precipitated using antibodies against EEF2 protein (ABclonal Technology, Wuhan, China), and the purified protein and RNA were then analyzed according to the protocol in previous studies [18 (link), 19 (link)].

Water solubility (WS) was measured as follows. Films were cut into circumferences of 40 mm diameter and were immersed in 20 mL of distilled water with 2% (w/w) Tris-HCl pH 7.0 (Sigma-Aldrich). Samples were kept for 24 h at room temperature. After this time, the mixture was filtered using a vacuum pump and Whatman no 1 paper (Sigma-Aldrich) to recover the insolubilized pieces of films, and they were dried in an oven at 105 °C for 24 h. WS was calculated as follows [28 (link)]: $$\mathrm{WS}(\%)=\left(\mathrm{m}1-\mathrm{m}2\right)/\mathrm{m}1\times 100$$
where m1 is the weight (g) of the dry films, and m2 is the weight (g) of the solubilized and dry films. Experiments were carried out in triplicate and the reported results correspond to the mean value.