Piercetm bicinchoninic acid bca protein assay kit

Protein isolation and Western blot were carried out in the same manner as previously described [46 (link),47 (link),48 (link)]. Proteins from EVs were extracted with RIPA buffer (10 mM Tris, 150 nM NaCl, 1% deoxycholic acid, 1% Triton X, 0.1% SDS, and 1 mM EDTA) containing protease inhibitors (aprotinin, PMSF, and sodium orthovanadate). A Pierce^TM bicinchoninic acid (BCA) protein assay kit was used to assess protein content (Thermo Fisher Scientific, Waltham, MA, USA). A 4–20% Tris-Glycine gel (Bio-Rad, Hercules, CA, USA) was loaded with equal quantities of protein (20 g/lane) and electrophoresed under nonreducing conditions. Proteins were transferred to a PVDF membrane, blocked for 2 h in blocking buffer (PBS, 0.05 percent Tween20^®, 5% milk), and then probed overnight with the following primary antibodies: ALIX (Sc-166952; Santa Cruz, CA, USA); CD81 (Sc-7637; Santa Cruz); and GM130 (#12480; Cell Signaling, Danvers, MA, USA). The membranes were rinsed the next day and treated for 1 h at room temperature (RT) with the secondary antibodies donkey anti-mouse IRDye 800CW and donkey anti-rabbit IRDye 680RD (1:2000, LI-COR Biosciences, Lincoln, NE, USA). All antibodies were diluted according to the manufacturer’s instructions. A fluorescence scanner was used to photograph the membranes (Odyssey v.3.0, LI-COR Biosciences).

RIPA buffer (100 mg in 1 mL) was added to ileal and liver tissue samples milled in liquid nitrogen. After 20 min on ice, sample tubes were vortexed for 20 s, centrifuged, and protein concentrations were determined using the PierceTM bicinchoninic acid (BCA) Protein Assay kit (Thermo, USA) and 30 μg protein (4 μg/μl protein) used for western blot. Quantified proteins underwent 10% SDS-PAGE, were blotted to polyvinylidene fluoride membranes, incubated with 5% milk/TBST for 120 min at RT, and refrigerated for 12 h at 4 °C with VDR, GLP-1, and GAPDH primary antibodies. The next day, membranes were subjected to IRDye whole IgG secondary antibodies. Protein bands were processed using a Licor Odyssey Scanner. The loading control was GAPDH.

Immediately post mortem, a 20 cm segment of the spinal cord (L2–L4) was aseptically obtained after removal of the dorsal aspect of the lumbar vertebrae dorsal laminae. Dura mater and arachnoids meninges were gently dissected and washed with phosphate-buffered saline (PBS). Samples were sectioned and snap-frozen in liquid nitrogen and transported to the laboratory for further processing. Spinal cord segments of approximately 250 mg were homogenated in 1 mL of PBS using an Ultra Turrax tissue homogenizer (T10, IKA^®, Staufen, Germany) at 16,000 rpm three times for 30 s each at 4 °C. Samples were then centrifuged at 2000 g for 3 min, and the supernatant was collected. Protein quantification was performed using the Pierce^TM bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rochford, USA).

The levels of oxidative stress were determined by assessing the extent of lipid peroxidation by measuring the levels of malondialdehyde (MDA) using the thiobarbituric acid reactive substances (TBARS) assay kit (Cayman Chemical, Michigan, USA). The levels of total protein were quantitated by using the Pierce^TM bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc, Minnesota, USA) according to the manufacturer's manual. In brief, the frozen sciatic nerve was homogenized in 250 µL of RIPA buffer with PIC and centrifuged at 1600 g for 15 minutes at 4°C; the supernatant was collected and used for the measurement of the levels of MDA and total protein. For measurement of MDA levels, the supernatant was stimulated by the addition of sodium dodecyl sulfate solution. The reactions were terminated by adding acetic acid solution and thiobarbituric acid reactive substance reagent. After boiling for 1 hour, the samples were cooled on ice for 30 minutes and centrifuged at 1600 g for 10 minutes at 4°C. The samples were collected and were used to measure the MDA levels at 535 nm using Spectra Max i3 spectrophotometer (Molecular Devices LLC., Tokyo, Japan). The levels of MDA were determined using the standard curve and were calculated as nmol/mg total protein content.

The Caspase 3/cysteine protease P32 (CPP32) Colorimetric Assay Kit (BioVision, Inc., Milpitas, CA, USA) was used. Trypsinized cells were resuspended in ice-cold cell lysis buffer (stored on ice, 10 min). Cytosolic extract was obtained by centrifugation (10,000× g, 10 min, 4 °C). Protein determination was performed using the Pierce^TM bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein (100 µg) was diluted in cell lysis buffer, to which an equal volume of 2× reaction buffer containing 10 mM dithiothreitol (DTT) was added. Aspartate-glutamate-valine-aspartate (DEVD)-p-nitroaniline (pNA) (4 mM) was subsequently added (37 °C, 2 h, protected from light). Absorbance was measured at 405 nm using an ELx800 Absorbance Reader (BioTek Instruments, Inc., Winooski, VT, USA).

The tissues were homogenized and sonicated in ice-cold RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitors. After centrifugation, the supernatant was collected and protein concentration was determined using a Pierce^TM bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The protein lysates were electrophoresed on polyacrylamide gels and were transferred to polyvinylidene difluoride membranes. After blocking in 5% skim milk, the membranes were incubated with primary antibodies against caspase-3, cleaved caspase-3, cyclooxygenase-2 (COX-2) (Cell Signaling Technology, Danvers, MA, USA); 4-HNE (Abcam, Cambridge, UK), and β-actin (Sigma-Aldrich, Louis, MO, USA). Then, the membranes were incubated with appropriated horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA) followed by ECL detection (Bio-Rad, Hercules, CA, USA). Relative protein levels were normalized to those of β-actin and quantified using the ChemiDoc XRS + System (Bio-Rad, Hercules, CA, USA).

Total protein content in cannabis plant extracts was measured using the PierceTM bicinchoninic acid (BCA) Protein Assay kit (Thermo Fisher Scientific), as per the manufacturer’s instructions. The serial dilution of cannabis protein samples and protein standards (generated with bovine serum albumin, BSA) were plated onto a 96-well plate, mixed with BCA working reagent solution mix and incubated at 37 °C for 30 min. Following incubation, plates were cooled briefly at room temperature (RT) and colorimetric absorbance signal was measured at 560 nm using a spectrophotometer. A standard curve was generated using absorbance measures at 560 nm for BSA protein standards. Absorbance signals measured for cannabis protein extracts was plotted on the standard curve to estimate protein concentrations.