Micro bcatm protein assay kit

Immunoblotting was performed essentially as described (Győri et al., 2014 (link); Csete et al., 2019 (link)). Cells grown on 6-well tissue culture plates were washed with ice-cold PBS and lysed using radioimmunoprecipitation assay buffer (RIPA, containing 1% Triton X, 0.1% SDS, 0.5% sodium deoxycholate, 30 mM HEPES, 5 mM Na-EGTA, 10 mM benzamidine, and 20 mM sodium fluoride in physiological saline) supplemented with sodium-orthovanadate, phosphatase inhibitor cocktails 1 and 2, PMSF and aprotinin (all from Sigma). Cell debris was removed by centrifugation at 16,100 g. Protein concentration was determined using the Micro BCA^TM Protein Assay Kit (Thermo Fisher Scientific). Samples then were mixed with 4× reducing sample buffer and boiled for 10 min. Ten micrograms of total protein was run on a 14% SDS-polyacrylamide gel, electroblotted onto nitrocellulose membranes and stained with Ponceau. Membranes were then blocked with 3% dry milk in PBS and 0.1% Tween 20 (PBS-Tween), followed by immunoblotting with primary antibodies against eGFP (1:2,000; clone F56-6A1.2.3; Abcam) or β-actin (1:10,000; Clone AC-74; Sigma) diluted in 3% BSA in PBS-Tween, followed by peroxidase-labeled anti-mouse IgG antibodies (1:5,000; GE Healthcare) diluted in 3% dry milk in PBS-Tween. Signal was developed by ECL (GE Healthcare) and exposed to X-ray film.

The vesicle pellet obtained by use of the Total exosome isolation (TEI) reagent was lysed in 50 μl of RIPA buffer supplemented with complete protease-inhibitors (Roche Diagnostics, Mannheim, Germany) and PhosSTOP phosphatase-inhibitors (Roche Diagnostics). Lysates were sonicated twice (15 s) in a 4°C water bath. Protein concentrations were quantified using the Micro BCA^TM Protein assay kit (Thermo Fisher Scientific) according to manufacturer’s instructions. Twenty five microgram of vesicles extracted from plasma or CM were for western blotting. Protein expression was assessed using antibodies against CD63, TSG101 (both 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), β-actin, vimentin (1:5000 and 1:500 respectively, Sigma–Aldrich, St.Louis, MO, USA), collagen type I (1:1000, Abcam, Cambridge, UK), and PDGFRβ (1:1000, GeneTex, Irvine, CA, USA). Calreticulin (1:1000, Cell Signaling Technology, Danvers, MA, USA) was used to verify absence of cell contamination. As positive control, a protein cell lysate of the HepG2 cell line was used.

The left hemispheres (probe-free) of the rat brains subjected to microdialysis experiments were used for Western blots. The NAc was identified and dissected. The pieces of tissue were homogenized in 300 μL of a lysis solution (400 μL RIPA buffer (Millipore, Burlington, MA, USA), 400 μL Complete Mini protease inhibitor (Roche Diagnostics, Mannheim, Germany), 266.7 μL NaF, and 51.2 μL Na3VO4). Samples were sonicated (Kontes micro ultrasonic cell disrupter, KT50) 3 times for 10 s. Subsequently, the samples were shaken at 4 °C for 20 min. Finally, the tissues were centrifuged at 4 °C for 30 min at 15,000 RPM, and the supernatant recovered. Proteins were stored at −80 °C until quantification with the micro-BCATM Protein Assay Kit (Thermo Scientific, Pierce, Madrid, Spain) using an Epoch™ microplate spectrophotometer (BioTek^®, Winooski, VT, USA).

Twenty recombinant fluorescent proteins were produced in bacteria. These proteins are described in Supplementary Table S2 (see page related to fluorescent proteins). Coding inserts were amplified by PCR using the primers listed in Supplementary Table S1 and then cloned into the pET.15b vector (Novagen, 69661), resulting in the expression of N-terminal 12xHis tagged fluorescent (GFP or mCherry) recombinant proteins. Proteins were expressed in Escherichia coli BL21-pLys cells (Promega, L1195) after induction with 1 mM IPTG (Invitrogen, 15529019) overnight at 20°C. Purification of His-tagged fusion proteins was performed using Ni-NTA beads as described previously (34 (link)). Elution of absorbed proteins was performed with 50 mM Tris–Cl (pH 7.9), 500 mM NaCl and 250 mM imidazole. Purified proteins were stored at −80°C in 25% (v/v) glycerol. Protein purity was checked by Coomassie blue staining. Protein concentrations were determined by measuring absorbance at 280 nm (SimpliNano; Biochrom) and by performing a Bradford protein assay using a Micro BCATM protein assay kit (Thermo Fisher Scientific, 23235).

Dried CCGH and CCGH dispersed in DDW were imaged using a microscope (BZ-X710, KEYENCE Ltd., Osaka, Japan). The CDDP content of CCGH was evaluated. Herein, 5 mg of CCGH was placed in 1 mL of 0.01 M phosphate-buffered saline (PBS) containing collagenase D (100 μg/mL) and was left at 37 °C for 24 h for complete dissolution. The CDDP concentration in the dissolved solution was measured based on platinum detected by a polarized Zeeman Z-8000 atomic absorption spectrophotometer (Hitachi, Ltd., Tokyo, Japan).
The degradability of CCGH and the CDDP release from CCGH in vitro were evaluated. Herein, 7.5 mg of CCGH was added to 1.5 mL PBS, followed by reciprocal shaking at 60 strokes/min at 37 °C. At pre-determined time intervals (1, 6, 12, 13, 15, 18, 24, and 30 h), 200 μL of the supernatant was collected after centrifugation (5000 rpm, 4 °C, 10 min) and replaced with the same volume of PBS. After 12 h, PBS was replaced with PBS containing collagenase D so that the concentration of collagenase in the solution was 10 μg/mL. The degradability of CCGH was assessed by quantitating proteins in the supernatant with Micro BCATM Protein Assay Kit (Thermo Fisher Scientific, Inc.), and the CDDP release from CCGH was assessed by quantitating platinum in the supernatant with a polarized Zeeman Z-8000 atomic absorption spectrophotometer.

Cells were collected and then lysed using 8 M urea and 100 mM ammonium bicarbonate on ice. Lysate protein concentrations were measured by micro BCA^TM protein assay kit (Thermo Scientific, USA). After normalization, equal amounts of proteins diluted in 1x LDS loading buffer containing 0.1 M DTT were boiled for 10 minutes at 95°C prior to electrophoresis. After gel electrophoresis, proteins were then transferred onto a nitrocellulose membrane using iBlot 2 Dry Blotting System (Thermo Scientific, USA), blocked with 5% skim milk in 1x TBST for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies. Following membrane washing, secondary antibodies IRDye 800 goat-anti-rabbit (Li-COR Biosciences, USA) or IRDye 800 goat-anti-mouse (Li-COR Biosciences, USA) were added for 1 hour at room temperature and washed three times with 1x TBST. The blot was then imaged with the Odyssey Classic Imager (Li-COR Biosciences, USA).

The sample on the membrane was washed three times with 100 μL of 100 mM ABC (14000 g, 45 min, 25°C), and trypsin diluted in 50 μL of 100 mM ABC was added in a 1:40 enzyme:protein ratio. Following overnight digestion at 37°C, the digest was collected by centrifugation (14000 g, 10 min, 25°C), subjected to two additional washes with 50 μL of 100 mM ABC, then concentrated in the SpeedVac to ∼20 μL.
One microliter of NH₄OH was added to the sample, then 5 μL of the propionylation reagent prepared by mixing propionic anhydride with MeCN in a 1:3 ratio was added. The pH was adjusted to 8–9 by NH₄OH, the sample was incubated in thermomixer at 37°C at 700 rpm for 20 min, then the sample volume was reduced in the SpeedVac to 5 μL. For the second round of propionylation, the sample was diluted with 50% (v/v) MeCN to a volume of 20 μL. The second round of propionylation was carried out with the same protocol. The sample was diluted with 0.1% formic acid (FA) to a volume of 100 μL. Labeled histones were desalted using a HyperSep SpinTip C-18 column (Thermo Fisher Scientific), and the peptide concentration was determined using a Micro BCA^TM Protein Assay Kit.