Stably-transfected 293T cells producing mouse R-spondin1-Fc fusion protein was a kind gift from Calvin Kuo at Stanford University. This fusion protein contains a C-terminal mouse IgG2-Fc fragment and was purified from the conditioned medium of the R-spondin1-293T cells via protein A affinity chromatography [18 (link)]. Briefly, R-spondin1-293T cells were cultured in a Nunc Polystyrene Cell Factory System (Thermo Scientific, Waltham, MA, USA), and the conditioned medium was collected and filtered. The R-spondin1-Fc protein was then purified using protein A Ceramic HyperD® F Affinity Chromatography Sorbent (Pall, Port Washington, NY, USA), and concentrated in saline using the 10 K Amicon-Ultra Centrifugal Filter (Millipore, Billerica, MA, USA). The yield of the purified protein was determined with a BCA Protein Quantification Assay Kit (Beyotime, Nantong, China). The level of R-spondin1-Fc protein was verified by CBB staining and immunoblotting with the R-spondin1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA).
Bca protein quantification assay kit
Manufactured by Beyotime
Sourced in China
The BCA-protein quantification assay kit is a colorimetric method for determining the total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) reaction to measure the reduction of copper ions by proteins in an alkaline environment. The assay produces a purple-colored reaction, and the intensity of the color is proportional to the protein concentration, which can be measured spectrophotometrically.
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4 protocols using bca protein quantification assay kit
1
Purification of Mouse R-spondin1-Fc Fusion Protein
2
Western Blot Analysis of ATDC5 Cells
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The ATDC5 cells at the concentration of approximately 3 × 105 were seeded in 6-well plates. The cell density was reached 80% before transfected with plasmids. 48 h after transfection, cells cultured medium was removed and RIPA was added to extract protein. The concentration of protein was measured by BCA protein quantification assay kit (Beyotime, Shanghai). Then proteins samples were separated by SDS-PAGE (5% of concentration gel and 12% of separation gel) and then transferred to PVDF membranes. The membranes were blocked by 5% skimmed milk for 1.5 h and incubated with primary antibodies [DDX3X, P65, p-P65, IkappaB (IkB-α), p-IkappaB (p-IkB-α), MMP-3, MMP-13]at 1:1000 dilution overnight at 4 °C. After washing the bands for three times with TBST, the secondary antibody of goat anti-rabbit was used to incubated for 1 h at 1:10,000 dilution at room temperature. According to the manufacturer’s protocol, the protein membranes were detected with ECL immunoblotting kit (Millipore, USA).
3
Bronchoalveolar Lavage Fluid Analysis
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Bronchoalveolar lavage (BAL) was performed by tracheotomy and injecting 0.8 mL cold PBS through the trachea, followed by carefully withdrawing. The volume of retrieved fluid was >60% of the injected one. BAL cells were collected by centrifuging the BALF at 1200 rpm for 10 min at 4 °C and then resuspended in PBS solution. Total number of cells were counted on a hemocytometer with trypan blue exclusion. Aliquots of the BALF were cytocentrifuged onto a glass slide and stained with Wright-Giemsa (Baso Diagnostics, Inc. Zhuhai, China). The differential cell counts were determined by counting 200 cells under 400× magnification. Meanwhile, the BALF supernatants were stored in −80 °C freezer until further cytokine analysis. The release of keratinocyte chemoattractant (KC) and CCL2 in BALF were analyzed by ELISA kits (eBioscience) according to the manufacturer’s instructions. The BAL total protein was measured by BCA-protein quantification assay kit (Beyotime, Shanghai, China). BAL cells were fluorescently labelled with CD11b (clone M1/70, Biolegend) and F4-80 (clone BM8, Biolegend) antibodies, and CD11b+F4-80+cells (macrophages) were sorted out by Flow for the uptake analysis in BAL macrophages by ICP-MS.
4
Quantification of Metabolic Markers in Mice
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Calcium concentrations in mouse plasma, liver and epididymal fat were determined by the colorimetric method with the calcium detection kit (cat. no. C004-1, Nanjing Jiancheng Bioengineering Institute, China). Plasma and hepatic TG and TC concentrations were assayed by an an enzymatic procedure with a TG assay kit by GPO-PAP method and a TC assay kit by COD-CE-PAP method respectively (Maccura Biotechnology Co., Ltd, Sichuan, China). The BCA-protein quantification assay kit (P0010S, Beyotime Institute of Biotechnology, Shanghai, China) was used to measure protein contents in hepatic tissues. The data were expressed as mmol/g protein for the liver and mmol/L for the plasma.
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