Total protein loading was determined by complete dissolution of the particles in dimethyl sulfoxide (DMSO; American Bioanalytical, Natick, MA). The DMSO-polymer solution was then diluted 1:15 in 2.5% (w/v) sodium dodecyl sulfate (Sigma Aldrich)+0.1N sodium hydroxide. The mixture was thoroughly vortexed prior to completing a micro bicinchoninic assay (BCA; G Biosciences; St. Louis, MO) to quantify protein using manufacturer’s protocols. Standards were prepared with known amounts of soluble BSA supplemented with blank PLGA particles (that is, no protein encapsulated, but produced using identical formulation/fractioning protocols). Encapsulation efficiency (EE) was calculated based on the ratio of total protein measured versus the total protein added during fabrication.
Bicinchoninic acid (bca)
Manufactured by G Biosciences
Sourced in United States
The BCA (Bicinchoninic Acid) is a colorimetric assay for the quantitation of total protein. It utilizes the reduction of Cu2+ to Cu+ by protein in an alkaline medium, and the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) using a reagent containing bicinchoninic acid.
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Lab products found in correlation
3 protocols using bicinchoninic acid (bca)
1
Quantifying Protein Encapsulation in Polymeric Particles
2
Whole Cell Lysate Preparation for p73 Analysis
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To prepare whole cell lysates, cells were collected by centrifugation and washed once with PBS and then lysed in EBC lysis buffer (Cold Spring Harb Protoc; 2006; doi:10.1101/pdb.rec8856) [50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40] supplemented with aprotinin (11.5 μg/ml), leupeptin (11.5 μg/ml) phenylmethylsulfonyl fluoride (50 μg/ml), NaF (100 mM) and Na ortovanadate (0.2 mM). Protein concentration was determined by BCA following the manufacturer's instructions (G Biosciences). Proteins (25 μg) were resolved in SDS/PAGE and transferred to PVDF filters. Blots were incubated with a mouse antibody against p73 (ER-15) (Thermo-Pierce) and then incubated with an anti-mouse antibody conjugated with horseradish peroxidase (Santa Cruz). Bound antibody was detected by a chemiluminescence assay (GE Healthcare).
3
Quantification of Cytokine Profiles
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Briefly, protein concentration of each sample was determined using bicinchoninic acid (BCA; G-Biosciences, St. Louis, MO, USA) and equal concentration of protein was used for the assay. Then, 25 µL of each sample and cytokine standards of different concentration were added to a 96-well plate. Further, samples were checked for the concentrations of the cytokines (IL-6, IL-8, IL-10, IL-17, IL-1β, and TNF-α) by MILLIPLEX enzyme-linked immunosorbent assay (ELISA) using MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A (HCYTA-60K-08; Millipore Sigma, Burlington, MA, USA). The experiment was conducted according to manufacturer's instructions. After incubation with primary and secondary antibodies, the plate was washed rigorously with wash buffer and the plate was resuspended in drive fluid. The plate was read and analyzed in the Luminex MAGPIX Multiplex System (Merck Millipore). Standard curves of known concentrations of recombinant human cytokines (R&D Systems, Minneapolis, MN, USA) were used to convert fluorescence units to cytokine concentration (pg/mL).34 (link)
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