Effective Inhibition of Foam Cells Formation by Tanshinone IIA in RAW264.7 Macrophages Induced with LDL Isolated from Hypercholesterolemia Patients: A Proteomic Analysis

Biochemical Studies on Cerebrospinal Fluid in Patients With Unresponsive Wakefulness Syndrome: Toward a Potential Search for Biomarkers

“…Two-dimensional electrophoresis was performed as previously described [17]. Brie y, 400 µg of protein sample from CSF was diluted with a hydration solution (8 mol/L urea, 13 mmol/L DTT, 0.5% IPG buffer, 2% CHAPS, 0.002% bromophenol blue) to a total volume of 450 µl and loaded on the IPG Ready Strip (Bio-rad, USA) with the method of in-gel swelling, with two replicates per sample.…”

“…At the end of electrophoresis, the gels were transferred to a staining box for silver staining. The obtained electrophoretograms of different samples were scanned using a UMAX Image Scanner (GE Healthcare, UK) at 300 pixels per square inch, and then analyzed using Image Master 2D Plantinum 6.0, software (GE Healthcare, UK) for point identi cation, background elimination, point matching, and analysis of differential proteins as described [17]. Each sample was analyzed in triplicate to reduce experimental errors.…”

“…Next, the spots were dehydrated with acetonitrile after washing twice with Milli-Q water and then dried in a SpeedVac (Eppendorf, USA) for 30 min. Subsequently, the spots were rehydrated with 5 µl trypsin solution (20 ng/µl in 200 mM NH 4 HCO 3 ) and incubated at 37 ℃ for 12 h. Finally, after washing twice with 30 µl of 50% acetonitrile and 5% tri uoroacetic acid, 1 µl of the peptide mixture was mixed with an equal volume of cyano-4hydroxycinnamic acid (10 mg/ml; Sigma, USA) saturated with 50% acetonitrile in 0.05% tri uoroacetic acid, and detected by a matrix-assisted laser desorption/ionization time of light matrix-assisted laser desorption/ionization time of ight mass spectrometry (MALDI-TOF-Mass Spectrometer) (4800 Proteomics Analyzer, Applied Biosystems, USA) as described [17].…”