Protein carbonyl elisa kit

The bioactive substances present in the culture supernatant were analyzed using the TGF-β1 ELISA kit (#KE00002, Protein Tech, Rosemont, IL, USA), IL-8 ELISA kit (#KE00006, Protein Tech), and Sonic hedgehog (SHH) ELISA kit (#ab100639, Abcam) according to the manufacturer's protocol. The samples were analyzed on day 3 or day 4 when ballooning was first observed (Fig. S2). On day 11, PHH/HSC sheets were homogenized, and protein lysates were prepared. Lysates were analyzed using the p62 ELISA kit (#ADI-900-212, ENZO New York, NY, USA), and protein carbonyl ELISA kit (#STA-310, CELL BIOLABS, San Diego, CA, USA) according to the manufacturer's protocol. The BCA protein assay kit (#T9300A, Takara Bio) was used to determine the protein concentration in the lysates. The Human Albumin ELISA kit (#E88-129, Bethyl Laboratories, Inc., Waltham, MA, USA) and Urea assay kit (#DIUR-100, BioAssay Systems, Hayward, CA, USA) were used to assess hepatocyte function. The samples were analyzed on days 0, 1, 5, and 10.

Total proteins were extracted from about 10⁸ yeast cells as described in paragraph 2.8. Protein carbonyl content was determined using Protein Carbonyl ELISA kit following the manufacturer’s instructions (Cell BioLabs, Paris, France).

The intracellular level of protein carbonyls was measured immediately after CS exposure (acute) and at T1, T12, as well as PT20. Protein carbonyls were quantified using a Protein Carbonyl ELISA kit and by following the manufacturer’s instructions (CellBio Labs, San Diego, CA) as described previously (Xiong et al. 2018 (link)). Briefly, ELISA plates were coated with 100 µL standards or 1 µg cell lysate diluted in 100 µL Dulbecco’s phosphate-buffered saline (DPBS) at 4 ℃ overnight. Carbonyl groups were derivatized with an equal volume of 0.04 mg/mL 2,4-dinitrophenylhydrazine (DNPH) for 45 min at room temperature. Derivatized proteins were detected using an anti-DNP antibody and a HRP-conjugated secondary antibody, followed by color development using the kit-provided 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate. The absorbance was measured at 450 nm using a BioTek Synergy H4 microplate reader. Reduced bovine serum albumin (BSA) standard was used as the background control.

The membrane lipid peroxide was measured using thiobarbituric acid (TBA; Sigma Aldrich, Saint-Quentin Fallavier, France). Approximately 10⁸ yeast cells were crushed in double distilled water and centrifuged at 10,000× g for 10 min. The supernatant (75 µL) was combined with 25 µL of SDS (3% w/v), 50 L of TBA (3% w/v) produced in 50 mM NaOH, and 50 µL of HCl (23% v/v). Between each addition, the mixture was thoroughly mixed. The resulting combination was heated to 80 °C for 20 min, then cooled on ice before measuring the absorbance at 532 nm (A532). Absorbance at 600 nm (i.e., non-specific absorbance measurement) was removed. A standard curve was prepared using 1,1,3,3, tetramethoxypropane to measure the concentrations of TBARS in the samples.
Total proteins were extracted from about 10⁸ yeast cells as described above (Section 4.11). The protein carbonyl content was determined using a Protein Carbonyl ELISA kit following the manufacturer’s instructions (Cell BioLabs, Paris, France).
DNA was extracted from about 10⁸ yeast cells with a Yeast DNA Extraction Reagent Kit following the manufacturer’s instructions (Thermo Scientific, Illkirch, France) and the 8-oxo-guanine content was determined with the 8-OHdG DNA Damage ELISA kit following the manufacturer’s instructions (Cell BioLabs, Paris, France).

The total proteins were extracted from about 10⁸ yeast cells following the method given in Section 2.12 The protein carbonyl content was determined using the Protein Carbonyl ELISA kit according to the manufacturer’s instructions (Cell BioLabs, Paris, France).