Pierce bca protein kit

Cells were homogenized on ice in RIPA buffer (Thermo Scientific) and supplemented with Halt Protease and Phosphatase Inhibitor cocktail (Thermo Scientific). Protein concentration was determined using the Pierce BCA protein Kit (Thermo Scientific). Primary antibodies included anti-cleaved PARP (Cell Signaling), anti-Caspase-3 (Cell Signaling), anti-ATG5 (Novus), anti-ATG7 (Cell Signaling), anti-LC3 (Cell Signaling), and anti-Cathepsin L (Santa Cruz). Signals were detected with HRP-conjugated secondary antibodies and chemiluminescence substrate (ECL, Millipore). Equivalent loading was confirmed with anti-β-actin (Sigma), anti- β-tubulin (Sigma), and anti-SHDB (Sigma) (for lysosome/mitochondria rich fractions).

The indirect quantification of proteins was performed using the Pierce BCA Protein Kit (Thermo Scientific—MA, USA). NTA was performed using Nanosight NS300 with the following parameters: 38.5 °C, capture of 30 s, and 5 reads.
Western blot was performed to detect the exosome marker CD63; the negative control was cytochrome c (Santa Cruz Biotechnology, Inc.—Texas, USA). This step was performed jointly with another study from our group that has already been published [52 (link)]. Briefly, 4× Laemmli buffer and mercaptoethanol (1:10) were used for protein extraction (5 min at 95 °C). The protein concentration was quantified with the Pierce BCA Assay kit and 5 µg of protein was added to each lane of a polyacrylamide gel (separation gel 12%, stacking gel 4%). The protein was separated at 100 V for 140 min. The separated protein was transferred to a nitrocellulose membrane at 80 V for 120 min. The nitrocellulose membrane was incubated with a solution that contained the primary antibody in 1× Tris-buffered saline with Tween 20 (TBS-T) and 1% bovine serum albumin (BSA) overnight at 4 °C. Then, the membrane was incubated in a solution that contained the secondary antibody in 1× TBS-T 1× and 5% BSA. The data were analyzed with ImageQuant LAS 4000 software version 1.2.

Western blotting was performed after the behavioral experiment, and behavioral and expression data were analyzed for each animal individually. Total proteins were extracted from the olfactory bulb tissues using RIPA lysis buffer (Solarbio, Beijing, China), and the protein concentrations were measured using a Pierce™ BCA protein kit (23227, Thermo Fisher Scientific, Waltham, MA, United States). Equal amounts of protein lysates (approximately 40 μg) were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States). The membranes were incubated with 5% BSA to block non-specific binding. Next, the membranes were probed with specific primary monoclonal antibodies at 4°C overnight, including anti-α-syn (SC-7011, 1:3000, Santa Cruz Biotechnology) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab9485, 1:3000, Abcam). Subsequently, the membranes were incubated with HRP-labeled anti-rabbit IgG (ab288151, Abcam) at 37°C for 2 h. Finally, protein expression was visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific). GAPDH was used as an internal reference for normalization.

Two-milliliter Tough Tubes with caps, ceramic beads 1.4 mm and 2.8 mm, and Omni Bead Ruptor 24 were purchased from Omni International, Kennesaw, GA, USA. Pipette tips (200 µL Genomic LR (orifice diameter 1.5) low retention) were purchased from VWR International, Radnor, PA, USA. Data were integrated with Lab Solutions 5.97 Shimadzu Corporation, Kyoto, Japan, Lab Solutions Insight LCMS. Pierce™ BCA Protein Kit was obtained by Thermo Scientific, Waltham, MA, USA. LC-MS grade isopropanol, methanol, and acetonitrile were obtained from Fischer Chemicals, Pittsburgh, PA, USA. Taurocholic acid, glycocholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and their corresponding deuterium-labeled compounds, as well as hydrochloric acid 37%, ammonium formate, and formic acid were purchased from Sigma-Aldrich, Burlington, MA, USA.