Pierce bca assay kit

For this experiment, metabolite extracts were prepared from liquid cultures. C. crescentus strains were grown at 30°C in the appropriate growth media until reaching OD₆₆₀ ~0.2–0.4 and ~1.5x10⁹ of cells were quickly collected onto a 0.45 μm PES membrane filter (Millipore) by vacuum filtration. The membrane filter was plunged into 0.5 M formic acid solution (kept at 4°C) to quench metabolism. Cells were washed off the membrane filters and vortexed briefly. The solution was kept at 4°C for 1 h. Lysates were clarified by centrifugation, frozen at -80°C and lyophilized. Metabolites were resuspended in 10 mM Tris-HCl pH 7.6, and KG was quantified using the KG assay kit (Sigma-Aldrich) following the manufacturer’s recommendation. The protein content in the extract was measured for normalization using the Pierce BCA assay kit (Thermo Fisher Scientific).

Tissue lysates were prepared from minced frozen samples of PDAC and adjacent normal pancreas on ice in a lysis buffer (50 mM Tris [pH 7.6], 0.5 M NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.02% NaN₃, 1 mM phenylmethanesulfonyl fluoride [PMSF], 5 mM ethylenediaminetetraacetic acid [EDTA], and 1 mM iodoacetamide) containing protease inhibitor cocktail (Roche, Basel, Switzerland), homogenized overnight at 4° C, and centrifuged at 10,000 g for 10 min at 4° C. Supernatants were kept frozen at −80° C until required. Protein concentrations of the lysates were determined using a Pierce BCA assay kit (Thermo Fisher Scientific, Rockford, USA).

The activity of MMP-2 and -9 was assessed by gelatin zymography as previously described [8 (link), 27 (link)]. Protein concentration was determined by Pierce™ BCA assay kit (Thermo Scientific, Rockford, IL). Murine and human BALF and lung homogenates were separated by electrophoresis using 10% SDS-polyacrylamide gels containing 0.1% gelatin. Gels were then washed in 2.5% triton 100 renaturing buffer followed by overnight incubation in developing buffer. To visualize bands gels were stained with 0.5% Coomassie blue for 1 h and then destained with 40% methanol/10% acetic acid until clear bands were visualized. Densitometry was performed as previously described and the MMPs activity were normalized for total BALF and lung homogenates protein concentration.

ARPE-19 cells were seeded in 6-well plates to confluence and serum starved for 2 days before adding TGFβ2 for up to 3 days. Cells were lysed in cold 1× Cell Lysis Buffer (Cell Signaling Technology) supplemented with 1 mM PMSF (Sigma). Protein concentration was quantified using the Pierce BCA Assay kit (Thermo Fisher). Citrate synthase activity was measured using the MitoCheck citrate synthase activity assay kit (Cayman Chemical, Ann Arbor, MI, USA) as per the manufacturer’s directions in a clear 96-well microplate. Absorbance was measured at 412 nm using a BioTek-Synergy 2 (BioTek Instruments, Inc., Winooski, VT, USA).

Cells were lysed in freshly prepared lysis buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% nonidet 40; 5 mM DTT; 10 μg aprotinin; 10 μg leupeptin; 10 mM PMSF; 10% glycerol) on ice, and centrifuged at 12,000 rpm for 15 min. The supernatant was saved as whole-cell lysate, and protein concentrations were measured using the Pierce BCA Assay Kit (Thermo Scientific). For immunoblotting, 50–100 μg of protein was separated on a 5% stacking and 8% separating SDS-PAGE gel. Protein was transferred in Tris-glycine buffer with 20% methanol onto nitrocellulose membranes, and blocked with 5% non-fat milk in TBST, pH 7.5. After blocking, membranes were incubated overnight at 4 °C with primary antibodies (polyclonal rabbit anti-STIL, 1:200; Santa Cruz Biotechnology; polyclonal rabbit anti-ACTIN, 1:3,000, Sigma; polyclonal rabbit anti-cleaved Caspase-3, 1:3,000, Abcam) diluted in fresh blocking solution. The membranes were washed with TBST, and then incubated for 1 hour at room temperature with secondary antibodies (goat anti-rabbit IgG conjugated with HRP, 1:3000 for STIL, 1:30,000 for ACTIN) diluted in fresh blocking solution. The membranes were washed with TBST, and protein expression was detected using the Super-Signal West Pico Chemiluminescent Substrate (Thermo Scientific).

The enzyme-linked immunosorbent assay was performed using the EBioscience assay Mouse IgG Total ELISA Ready-SET-Go! The protein concentration was determined using the Pierce BCA assay kit (Thermo Fisher Scientific).