Pierce bca protein assay kit

The ligation efficiency of plCSA-BP on DGL/CSA-PNPs was evaluated by a Pierce BCA Protein Assay Kit (Thermo Inc., Waltham, MA, USA) by following the instructions [43 (link)]. The unconjugated plCSA-BP or SCR peptide were removed by ultrafiltration, using a 5 kDa molecular weight cutoff membrane. Thereafter, plCSA-BP or SCR peptide concentration conjugated on DGL/CSA-PNPs and DGL/SCR-PNPs were evaluated by Pierce BCA Protein Assay Kit (Thermo Inc., Waltham, MA, USA). The morphological and surface characteristics of the DGL/CSA-PNPs were examined by transmission electron microscopy (Hitachi Ltd., Tokyo, Japan). Zeta potential and particle size of diameter of the nanoparticles were measured by Zetasizer Nano (Malvern, England) by following the manufacturers’ instructions. Encapsulation efficiency (EE) and loading capacity (LC) were calculated with the following equations:

where W₀ is the total amount of prodigiosin, W₁ represents the weight of free prodigiosin, and W_N indicates the weight of the nanoparticles. The amount of prodigiosin in the nanoparticles was measured by a Beckman Coulter (Indianapolis, IN, USA) DU 800 UV-Vis spectrophotometer, based on their absorbance at 533 nm.

For total protein extraction, co-cultured cells exposed to HS/hypoxia for 2 h were lysed immediately after removal from the incubator using 50 μl Pierce RIPA buffer (Thermo Fisher Scientific, USA) containing protease inhibitor cocktail (#11836170001, Roche, Switzerland). Total protein concentration was assessed and standardized with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The protein samples used for determining HIF-1α expression were preincubated with dimethyloxalylglycine (DMOG) (Sigma-Aldrich, USA), a HIF prolyl-hydroxylase inhibitor, which stabilizes HIF-1α during the normoxic lysis procedure.
Nuclear and cytoplasmic proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, USA) and standardized by using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA).

The protein concentrations of tumour tissue-derived EVs isolated by protocols 1 and 2, as well as for ELISA experiments were measured by using the Pierce BCA™ Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Protein concentrations in tumour tissue-derived EVs isolated by protocol 3 were valuated using the Pierce BCA™ Protein Assay Kit (Thermo Fisher Scientific) or the Qubit (Thermo Fisher Scientific) according to the manufacturer’s protocol.
The protein concentrations of the human mast cell line HMC-1, as well as the tumour tissue pellet obtained after 300 × g was evaluated by using RIPA buffer (Thermo Fisher Scientific) and protein inhibitors (cOmplete™, Mini Protease Inhibitor Cocktail, Roche) and was created to be used as a control for the Western blot experiments. An aliquot of the isolated EV samples was mixed with RIPA buffer and used for the Western blot analysis. The protein concentrations of samples used for Western blot experiments (cell lysate, 300 × g pellet and isolated EVs) were measured by Qubit (Thermo Fisher Scientific) according to the manufacturer’s protocol.
The protein concentrations of the EVs isolated from the density gradients were measured by Qubit (Thermo Fisher Scientific) according to the manufacturer’s protocol.

Stomachs and colons of ND lean or HFD obese rats were collected after euthanasia, washed to remove the debris and then cut into small pieces. The small pieces of each segment were incubated in a 37 °C bath with stirring in medium containing PBS continuously gassed with 95% O₂–5% CO₂. After a 30-min stabilization incubation, PBS (without IBMX) and 100 µM 3-isobutyl-1-methylxanthine (with IBMX) (Sigma Chemicals, St Louis Mo) was added and incubated for 60 min so that each rat served as its own control. Afterwards, the supernatants were collected and the fragments snapped frozen prior to homogenization and protein quantification with the Pierce BCA Protein Assay Kit (Prod #23225, ThermoFisher Scientific, France). The supernatants were stored at −20 °C until GLP-1 RIA. The GLP-1 release (pM) in each incubated fragment was normalized to the quantity of protein determined with the Pierce BCA Protein Assay Kit (Prod# 23225, ThermoFisher Scientific, France).

For HEK293 cells, cells were harvested and lysed in lysis buffer by pipetting up and down 50 times. Supernatants were collected after 5 min of centrifugation at 8000 × g, and protein concentration was determined by using Pierce BCA protein assay kit from Thermo Fisher before they were resuspended in sample buffer for Western blot analysis. For preparing tissue lysates from mouse livers and prostates, tissues were sliced into microcentrifuge tubes pre-aliquoted with 300 mL lysis buffer with protease inhibitors. Tissues were minced with a handhold tissue homogenizer for 1 minute before the microcentrifuge tubes were subjected to centrifugation at 8000 × g for 5 mins. The supernatant from each tube was collected for measurement of protein content using Pierce BCA protein assay kit from Thermo Fisher and for Western blot analysis.

BlCa cell lines, sh-scramble/CTRL and sh-SIRT7 cells were grown until 80% confluence and homogenized in Kinexus lysis buffer supplemented with proteases inhibitors cocktail. Then, cells were sonicated for 5 cycles of 30 s ON and 30 s OFF (Bioruptor^®, Diagenode, Liège, Belgium). After centrifugation, the supernatant was collected, and total protein was quantified according the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), according to the manufacture procedure.
For subcellular fractionation, Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) was used. Briefly, bladder cancer cell lines, sh-scramble/CTRL and sh-SIRT7 cells were washed in 1 X PBS with phosphate inhibitors and scrapped. Subsequently, cells were suspended in hypotonic buffer and incubated on ice during 15 min. Additionally, a detergent was added, and samples were centrifuged at 14,000 rpm during 30 s at 4 °C. Supernatant (cytoplasmic fraction) was collected and stored at −80 °C until use. Then, cell pellets were resuspended and incubated in a complete lysis buffer solution (lysis buffer with proteases inhibitor cocktail and 10 mM DTT), following centrifugation and supernatant (nuclear fraction) collection and storage at −80 °C. Nuclear and cytoplasmic proteins were then quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), according to manufacture procedure.

Cells were scraped on ice, pelleted down at 2000× g for 5 min and resuspended in pre-cooled RIPA buffer (150mM NaCl, 50mM Tris pH 8.0, 5mM EDTA pH 8.0, 1% NP-40, 0.5% sodium deocycholate, 0.1% SDS) containing proteinase inhibitor cocktail (Roche, Basel, Switzerland), vortexed and incubated on ice for 30 min with being vortexed every 10 min. The resuspension was finally centrifuged for 10 min at 10,000× g at 4 °C, the resulting supernatant was mixed with SDS sample buffer and heated to 95° for 5 min. Total protein concentration of the lysate was determined using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Darmstadt, Germany).
To prepare tissue lysates, 8-weeks old male C57BL/6 wild-type mice were sacrificed by cerebral dislocation. The mouse organs were homogenized with Triple-Pure High Impact Zirconium Beads with a 1.5 mm diameter in RIPA buffer using a BeadBug 6 Position Homogenizer (Benchmark Scientific, Edison, USA). To remove cell debris, the samples were centrifuged for 10 min at 1000× g at 4 °C (Heraeus Fresco 21, Thermo Fisher Scientific, Darmstadt, Germany). The supernatant was used as whole-organ lysate and the protein concentration was determined with the Pierce BCA Protein Assay kit (Thermo Fisher Scientific).