Dctm protein assay kit 2

Chickpeas (Cicer arietinum variety Kabuli), Yellow peas (Pisum sativum), Faba beans (Vicia faba), Red beans (Phaseolus vulgaris variety Kidney), green lentils (Lens culinaris) were generously donated by Pulse Canada (Manitoba, Canada). α-Glucosidase from Saccharomyces cerevisiae (≥100 units/mg protein), dipeptidyl peptidase IV human (recombinant expressed in Sf9 cells), α-amylase (from porcine pancreas, type VI-B, ≥5 units/mg solid), pancreatin (from porcine pancreas, 8xUSP specification), pepsin (from porcine gastric mucosa, ≥250 units/mg solid), Calcoflour white stain (calcofluor white M2R 1 g/L, evans blue, 0.5 g/L), Toluidine blue O, gallic acid, vanillin and catechin were purchased from Millipore Sigma (Burlington, MA, USA). Lactobacillus plantarum ATCC® 8014™ was purchased from Cedarlane (Burlington, ON, Canada). DeMan, Rogosa and Sharpe (MRS) broth, M17 broth and Bacteriological agar were purchased from Oxoid (Nepean, ON, Canada). DC^TM Protein Assay Kit II and Ladder Precision Plus Protein dual color standards were purchased from BioRad (Mississauga, ON, Canada). GelCode blue safe protein stain was purchased from Fisher Scientific (Toronto, ON, Canada).

Measurement of cellular respiration was performed using the Seahorse XF96e analyzer as previously described³² . Cells were grown in either KGM Gold or RM + medium and seeded out as described above. Prior to respiration assays, cell culture medium was replaced by prewarmed, pH 7.4 adjusted assay medium supplemented with 5 mM glucose, 10 mM sodium pyruvate and 2 mM glutamate according to the manufacturer’s protocol. Oxygen consumption rate (OCR), extracellular consumption rate (ECAR) and proton production rate were measured under basal conditions and in the presence of oligomycin, a complex V inhibitor (1 μM), the complex III inhibitor antimycin A (0.5 μM), the complex I inhibitor rotenone (0.5 μM) and the mitochondrial uncoupler carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1.5 μM) to assess maximal oxidative capacity. Following the respiration assay, media was removed, wells were washed once with PBS and total protein concentration was measured using the DCTM Protein Assay Kit II (BioRad). All cell lines were at least measured n = 10 times and normalized to total protein content, including a BSA standard with known protein concentrations. The data was later analysed using the Seahorse Report Generator. Spare respiratory capacity (SRC) was defined as the difference between basal and maximum respiration.

Pooled brain tissues were mechanically disrupted on ice in 200 μL RIPA extraction buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% nonyl phenoxylpolyethoxylethanol-40 (NP-40), 1% sodium deoxycholate and 0.1% SDS) (Pierce) containing a protease inhibitor tablet (proprietary formulation containing AEBSF HCl, aprotinin, bestatin, E-64, leupeptin, pepstatin, EDTA) (Pierce) and phosphatase inhibitor cocktail set I (2.5 mM bromotetramisole oxalate, 500 μM cantharidin, 500 nM microcystin-LR) and II (200 mM imidazole, 100 mM sodium fluoride, 115 mM sodium molybdate, 100 mM sodium orthovanadate, 400 mM tartrate dihydrate) (Calbiochem). Samples were clarified at 12,000 × g for 30 minutes at 15 °C and supernatants were transferred to 2 mL screw-cap tubes for precipitation. Samples were precipitated with 80% acetone/10% trichloroacetic acid (TFA) overnight at −20 °C. Precipitated proteins were pelleted by centrifugation at 20,000 × g for 30 minutes at 4 °C using an Eppendorf Centrifuge 5417 R, washed with 100% acetone, vortexed, and incubated on ice for 30 minutes. This was repeated with 80% acetone 2 × and then 80% ethanol. Washed, precipitated proteins were reconstituted in RIPA containing protease and phosphatase inhibitors. Total protein was determined by DC^TM protein assay kit II following the manufacturers protocol (Bio-Rad).