Fifteen microliters per lane was added to the gel and separated by SDS-PAGE (10C20% gradient gel), and then transferred to a polyvinylidene difluoridemembrane (pore size 0.2 m). extractions were not significantly different, but triglycerides were barely recovered from serum by methanol extraction, requiring the use of isopropanol. However, once the serum was enriched with CD9 magnetic beads, triglycerides, and phospholipids were recovered at similar levels in both methanol and isopropanol extractions. Therefore, it is possible that the triglyceride fraction of the whole serum and the triglyceride fraction were enriched in CD9 magnetic beads differ in localization and properties. In addition, the variation per disease was small in general serum lipidomics; however, the difference per disease appeared larger when CD9 magnetic bead enrichment was employed. for 5 min, before the supernatant was discarded. Next, 200 L of MeOH was added, the mixture was suspended well, it was centrifuged at 10,000 for 5 bio-THZ1 min, and the supernatant was discarded. Next, 200 L of 25 mM MES buffer (pH 6) was added to disperse the beads, which were centrifuged at 10,000 for 5 min before the supernatant was discarded. We then added 100 L each of 1 1 mg/mL CD9 antibody and IgG to Eppendorf tubes containing the magnetic beads. We added 100 L of 25 mM MES buffer (pH 6) and suspended the mixture. The mixture was agitated slowly at 4 C for 4 h. The mixture was then centrifuged at 10,000 for 5 min, and the supernatant was discarded. Subsequently, 300 L of a solution with final concentrations of 0.1 M HEPES buffer, 1 M ethanolamine, and 1% polyoxyethylene was adjusted to pH 8 with hydrochloric acid and added to the tube containing the magnetic beads, which was mixed slowly at 4 C overnight. The sample was centrifuged at 10,000 for 5 min, the supernatant was discarded and 300 L of 25 mM HEPES solution (pH 8) containing 2 mM EDTA was added. The magnetic beads were washed, centrifuged at 10,000 for 5 min, and the supernatant was discarded. Finally, we added 400 L of 25 mM HEPES solution (pH 8) containing 2 mM EDTA and stored the tube at 4 C. The protein immobilized bead concentration was approximately 0.1 mg/20 L. 4.4. Serum Enrichment with CD9 Antibody and IgG-Immobilized Magnetic Beads Then, 200 L of PBS was added to 100 L of serum, and 7 L of protein-immobilized magnetic beads were added while suspending them well. The mixture was slowly mixed for 2 h at 4 C. The water adhering to the back cover of the tube was also dropped into the tube, the magnetic beads were collected using a magnet, and the supernatant was discarded. After adding 500 L of PBS, the magnetic beads were mixed well, collected with a magnet, and discarded. Another 500 L of PBS was added and the magnetic beads were mixed well. After transfer to a new tube, the magnetic beads were collected using a magnet, and the supernatant was discarded. MeOH or IPA (50 L) was added and mixed, the magnetic beads were collected with a magnet, and the supernatant was analyzed by LC/MS. 4.5. Liquid Chromatography/Mass Spectrometry Measurements The LC and MS measurement bio-THZ1 conditions were as follows: Serum samples were subjected to a Nexera UHPLC system and LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Co., Kyoto, Japan). An Acquity UPLC BEH C8 column (1.7 m, 2.1 100 mm; Waters) was used with bio-THZ1 the following mobile phase compositions: 5 mM NH4HCO3/water (mobile phase A), acetonitrile (mobile phase B), and isopropanol (mobile phase C). The pump gradient was programmed as follows [time (%A/%B/%C)]: 0 min (95/5/0), to 8 min (70/30/0), 16 min (30/35/35), 28 min (6/47/47), 35 min (6/47/47), 35.1 min (95/5/0); it was then held for 38 min for equilibration. The flow rate was 0.35 mL/min, and the column temperature was 47 C. The injection volume was 5 L. SRM analysis was performed using positive/negative ion-switching mode ESI, with a collision energy of 46 eV. All data were analyzed using Microsoft Excel 2016. 4.6. Western Blot For serum-enriched fractions using the CD9 antibody and IgG-immobilized magnetic beads, 30 L of SDS-PAGE sample buffer was added after washing with PBS. Serum was diluted Rabbit Polyclonal to YOD1 50-fold with SDS-PAGE sample buffer because the of the excessive amount of protein. They were heated at 95 C for 3 min. Fifteen microliters per lane was added to the gel.