For whole cell protein extracts, cells were washed with 1x PBS and harvested using trypsin 0.25% EDTA. After centrifugation at 2,000 g for 3 minutes, the protein pellets were washed twice with ice-cold 1x PBS to remove any remaining FCS. Proteins were then extracted from the collected cells using 1 volume of whole cell extract buffer (50 mM Tris HCl pH 7.9, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 5 mM MgCl2, 600 mM KCl, 0.5% NP40 and 1x protein inhibitor cocktail) and incubated for 10 minutes. The salt concentration was neutralized by addition 2–3 volumes of IP0 buffer (25 mM Tris HCl pH 7.9, 5% glycerol, 5 mM MgCl2, 0.1% NP40, 1 mM DTT and 1x protein inhibitor cocktail) and incubation for 10 minutes. After centrifugation at 12,000 g for 10 minutes at 4°C, the supernatant containing the proteins was collected. The protein concentrations of the extracts were determined using the Coomassie Protein Assay Dye Reagent Concentrate (Bio-Rad, Cat#5000006) and Synergy HTX Multi-Mode Reader (BioTek).
Coomassie protein assay dye reagent concentrate
Manufactured by Bio-Rad
The Coomassie Protein Assay Dye Reagent Concentrate is a laboratory reagent used for the quantification of protein concentrations. It is a concentrated solution that is diluted and combined with samples to produce a colored complex, which can then be measured using a spectrophotometer to determine the protein content.
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2 protocols using coomassie protein assay dye reagent concentrate
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Whole Cell Protein Extraction Protocol
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Whole Cell Protein Extraction Protocol
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For whole cell protein extracts, cells were washed with 1x PBS and harvested using trypsin 0.25% EDTA. After centrifugation at 2,000 g for 3 minutes, the protein pellets were washed twice with ice-cold 1x PBS to remove any remaining FCS. Proteins were then extracted from the collected cells using 1 volume of whole cell extract buffer (50 mM Tris HCl pH 7.9, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 5 mM MgCl2, 600 mM KCl, 0.5% NP40 and 1x protein inhibitor cocktail) and incubated for 10 minutes. The salt concentration was neutralized by addition 2–3 volumes of IP0 buffer (25 mM Tris HCl pH 7.9, 5% glycerol, 5 mM MgCl2, 0.1% NP40, 1 mM DTT and 1x protein inhibitor cocktail) and incubation for 10 minutes. After centrifugation at 12,000 g for 10 minutes at 4°C, the supernatant containing the proteins was collected. The protein concentrations of the extracts were determined using the Coomassie Protein Assay Dye Reagent Concentrate (Bio-Rad, Cat#5000006) and Synergy HTX Multi-Mode Reader (BioTek).
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