Thermo bca kit

HCT116 and HCTR cells were seeded at a density of 0.25 × 10⁶ cells/mL in 1 mL medium in 12-well plates and left to recover overnight. The medium was replaced and the cells were treated with 100 µM BOLD-100 or the corresponding concentration of DMSO for 24 h. The cells were washed 3 times with PBS (pH 7.4), quenched with liquid nitrogen, and stored at −80 °C until further processing. The metabolomics experiments were carried out as described before [31 (link)]. In short, liquid chromatography high-resolution mass spectrometry (LC-MS) measurement with a Thermo Scientific Q Exactive HF quadrupole-Orbitrap mass spectrometer was utilized in full mass scan mode (both positive and negative ionization-mode) at a resolution of 120,000. External calibration with fully labelled ¹³C internal standards ISOtopic solutions (Vienna, Austria) was used for quantification. The obtained absolute metabolite amounts (pmol) were normalized to the total protein content in their respective well (µg) with the Thermo BCA kit, according to manufacturer’s instructions.

The total protein of tumor cells was extracted by RIPA buffer (89,901, Thermo Fisher Scientific), followed by the quantified procedure according to Thermo BCA kit (23,227, Thermo Fisher Scientific, USA). Total protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Germany). The membranes were blocked with 5% nonfat milk, followed by staining with primary antibodies. The primary antibodies included: rabbit anti-mouse Notch1 (ab52627, Abcam, 1:1000, UK) and rabbit anti-mouse GAPDH (AF1186, Beyotime, 1:5000, China). Secondary antibody was HRP-linked goat anti-rabbit IgG (SA00001-2, Proteintech, 1:2000, USA). Images were taken by Tanon-5200 Chemiluminescent Imaging System (Tanon, China).