The medium of the cells grown in 10 cm cell culture plate was removed, and the cells were washed once with PBS, and lysed with Pierce RIPA buffer (Theomo Scientific, Cat. # 89901) containing 1x Pierce protease and phosphatase inhibitors (Thermo Scientific, Cat. # 88668). The cell lysate was pelleted down at 15,200 xg at 4 °C for 15min, the cell supernatant was transferred to a fresh Eppendorf tube, and 5 uL of sample was quantitated by using (Quick Start Bradford Dyed Reagent, Biorad, Cat. # 500–0205). 50 μg of each protein sample was analyzed by SDS-PAGE (10% gel) followed by Western blotting, and autoradiography. Precision Plus Dual Color (Biorad, Cat. # 161–0374) was used as protein standard marker. Clarity Western ECL Substrate (Biorad, Cat. # 170–5060) was used to develop the probe signal, and Classic Blue BX film (MidSci, Ref. # 604 5983) was used for autoradiography.
Quick start bradford dyed reagent
Manufactured by Bio-Rad
The Quick Start Bradford Dyed Reagent is a pre-formulated solution for performing the Bradford protein assay. It is designed to quantify protein concentrations in samples.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using quick start bradford dyed reagent
1
Protein Extraction and Western Blotting
2
Macrophage Response to Interleukin-23 Signaling
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Briefly, human macrophage cell line (ATCC, CRL 9855) was treated with Phosphate-Buffered Saline (PBS, control) and Interleukin-23 (Biolegend). The cells were harvested at various time points, washed once with PBS, lysed with RIPA buffer containing 1× protease and phosphatase inhibitors, protein samples quantitated (Quick Start Bradford Dyed Reagent, Biorad), and 100 micrograms each of protein samples were analyzed by SDS-PAGE (10% gel) followed by Western blotting, and autoradiography. Precision Plus Dual Color (Biorad) was used as protein standard marker. TGFβ1 antibody (Novus, rabbit polyclonal), IL23 p19 antibody (ThermoFisher, rabbit polyclonal) β-actin antibody (Cell Signaling Technology, mouse mAb), anti-rabbit-HRP and anti-mouse-HRP (Cell Signaling Technology) were all used at 1:1000 dilution by following manufacturer’s instructions. Clarity Western ECL Substrate (Biorad) was used to develop the probe signal, and Classic Blue BX film (MidSci) was used for autoradiography.
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