Mp fastprep 24 automated homogenizer

The tissue was cut and an appropriate amount of SDT lysate was added. An MP FastPrep-24 Automated Homogenizer was used to crush and homogenize liver tissue (24 × 2, 6.0 M/s, 60 s, twice). After boiling (10 minutes), the tissue homogenate was centrifuged at 14,000 × g for 15 minutes. Subsequently, the supernatant was taken and filtered using a 0.22 µm centrifuge tube to collect the filtrate. After quantification and enzymatic hydrolysis, the peptide segment was desalinated using a C₁₈ cartridge. The peptide mixture of all samples was taken and subjected to high-PH RP grading using the Agilent 1260 infinity II HPLC system. Components were collected, and each was dried in a vacuum concentrator. After freeze-drying, the sample was redissolved with 0.1% formic acid aqueous solution and combined into six fractions. After tandem mass spectrometry analysis, the original mass spectrometry data were merged and analyzed using Spectronaut Pulsar X (version 12, Biognosys AG) to establish a spectrogram database.

SDT buffer (4% SDS, 100 mM Tris-HCl, pH 7.6) was added to the lysed liver, and transferred to 2-mL tubes. The lysate was homogenized by an MP Fastprep-24 Automated Homogenizer (6.0 M/S, 30 s, twice) (MP Biomedicals Inc., USA). The homogenate was sonicated and boiled for 15 min. After centrifugation at 14,000 g for 40 min, the supernatant was filtered using a 0.22-μm filter. The filtrate was quantified using the BCA Protein Assay Kit (P0012, Beyotime) [22 (link)].

The sample was transferred to 2 ml tubes with quartz sand and diluted with SDT buffer. Using the MP Fastprep-24 Automated Homogenizer, the lysate was homogenized twice at 6.0M/S for 30 seconds each time. A sonicator was used to homogenize the material, and then a 10-minute boil was performed. In addition to centrifuging at 14000g for 15 minutes, the supernatant was filtered through 0.22 μm filters. BCA Protein Assay Kit (P0012, Beyotime) was used to quantify the filtrate. Samples were stored at -80°C.