Quick start bsa standard set

Protein samples were prepared by lysing 20 mg of powdered tissue in the buffer: 50 mM Tris-HCl pH 7,8, 150 mM NaCl, 1% SDS, 1 mM EDTA, and protease inhibitor cocktail (Roche, Cat #1873580). The lysate was clarified by centrifugation at 14,000 g for 15 min. Protein concentration was measured using Quick Start Bradford 1x Dye Reagent (Bio-Rad, Cat #500–0205) and QuickStart BSA Standard Set (Bio-Rad, Cat #500–0207), readings were done on CLARIOstar Plus (BMG Labtech). Each protein lysate (10 μg or 5 μg for the abundance) was resolved in 12% SDS-polyacrylamide electrophoresis gel and transferred to a nitrocellulose membrane using the Trans-Blot Turbo Blotting System (Bio-Rad). The membrane was blocked in 5% dry milk in TBS-T and incubated for 1 h at room temperature with antibodies diluted in blocking solution: anti-Gαo (rabbit; Thermo Fisher Scientific, PA5-30044; 1:5,000 dilution), anti-GAPDH (mouse; Sigma-Aldrich, G8795; 1:20,000), anti-beta III Tubulin (rabbit; Abcam, ab18207; 1:1,000). Following washes in TBS-T the appropriate HRP-conjugated secondary antibodies were used: anti-rabbit (Bio-Rad, Cat# 170–6515; 1:3,000) and anti-mouse (Bio-Rad, Cat# 170–6516; 1:3,000). Proteins were detected using Clarity Western ECL substrate (BioRad, #170–5,060) and iBright 1500 Imaging System (Thermo Fisher Scientific).

ucMGP concentrations in GGCX dermal fibroblasts were quantified using a human ucMGP ELISA Kit according to the manufacturer’s protocol (#CSB-EC013789HU, Cusabio Biotech Co., Wuhan, China). Briefly, cells (6.4 × 10⁵) isolated from three controls and one GGCX patient were separately seeded on 6-well plates. When they reached early confluency, cells were washed with cold PBS twice and lysed on ice for 5 minutes with RIPA buffer (#9806S, Cell Signaling Technology) containing protease inhibitors (Protease Inhibitor Cocktail Set I; Calbiochem) and 1 mM phenylmethylsulfonyl fluoride (Santa Cruz). The lysate were collected using a cell scraper and vortexed for 30 seconds. After centrifugation at 12,500 rpm for 10 minutes at 4°C, protein concentrations in the supernatants were quantitated using a Quick Start Bradford protein assay (Quick Start BSA standard set, Bio-Rad Laboratories, Inc.). Samples (approximately 40 μg of protein) were mixed with the sample diluent, and the ucMGP content was assessed. Reactions were stopped by addition of acidic stop solution, followed by measurement of OD at 450 nm in triplicate (Labsystems Multiskan MS #352, Thermo Fisher Scientific Inc.). The values were adjusted by the protein contents, which were determined from the same plates.