The total protein concentration in the tested samples was measured using the colorimetric BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The assay was performed in quadruplicates, with the same supernatant samples used for the Luminex analysis (originating from 6 donors). For this analysis, samples were diluted 10× with sterile ultrapure water, to obtain the final concentration of protein in the sample in the range from 20 to 2000 µg/mL (linear working range for BSA). The absorbance was measured at 562 nm on a plate reader (PowerWave XS, BioTek, Santa Clara, CA, USA). To determine the protein concentration of each sample, the standard curve was plotted.
Colorimetric bca protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Colorimetric BCA Protein Assay Kit is a laboratory tool used for the quantitative determination of protein concentration. It employs the bicinchoninic acid (BCA) method to produce a purple-colored reaction product. The absorbance of this product is measured at a specific wavelength, allowing for the estimation of the protein content in a sample.
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3 protocols using colorimetric bca protein assay kit
1
Quantitative Protein Determination by BCA Assay
2
Rat Liver Protein Quantification
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The total protein content in rat liver homogenates was measured according to the manufacturer instructions using a colorimetric BCA protein assay kit (Cat. #23227, Thermo-Fisher Scientific® (Vienna, Austria).
3
Quantitative Western Blot Analysis of Liver Proteins
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Protein levels were analyzed by Western blot as described previously (Granzow et al., 2014 (link); Brol et al., 2019 (link)). Briefly, snap-frozen livers were homogenized and diluted. Protein quantification was performed using a colorimetric BCA protein assay kit (Cat 23225, Thermo Fisher Scientific Inc., IL, United States). Forty micrograms of protein samples was subjected to SDS-PAGE under reducing conditions (10% gels), and proteins were blotted on nitrocellulose membranes. The membranes were blocked and incubated with primary antibody against collagen 1α1 (SC-12895; Santa Cruz Biotechnology, Santa Cruz, CA), srebp1c (ab28481, Abcam), fas (ab82419, Abcam), caspase-1 (sc-56036, Santa Cruz Biotechnology, Santa Cruz, CA), IL-1β (NB600-633). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an endogenous control (sc-47724; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were incubated with the corresponding secondary antibody, and blots were developed using enhanced chemiluminescence. Protein quantification was performed by ImageJ (version 1.51q, NIH, United States) and results were corrected for GAPDH levels.
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