Total protein was extracted with the P-PER Plant Protein Extraction Kit (Pierce Chemical Co., Rockford, IL) following the manufacturer’s recommendations, with addition of cOmplete protease inhibitor cocktail (1X) (Roche, Mannheim, Germany) to the P-PER kit working solution, at 1:100 proportion. Total protein concentration was determined by the Bradford (1976 (link)) method, using bovine serum albumin (BSA) as a standard (more detailed information in Electronic Supplementary Material 1 [file ESM1.pdf]).
Complete protease inhibitor cocktail 1x
Manufactured by Roche
Sourced in Switzerland
The Complete Protease Inhibitor Cocktail (1X) is a laboratory reagent designed to inhibit a broad range of proteases. It is a pre-formulated mixture of protease inhibitors that can be added to protein samples to prevent degradation during extraction, purification, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using complete protease inhibitor cocktail 1x
1
Total Protein Extraction from Plants
2
Preparation of Normal Brain Homogenates
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Animals were sacrificed in strict accordance with the ethical standards of French and European laws (European Community Council Directive 2010/63/EU of 22 September 2010).
Ten percent (wt/vol) normal brain homogenates (NBH) were prepared in conversion buffer (PBS with 150 mM NaCl, 1% Triton X-100, 0.005% EDTA pH 8, and Complete Protease Inhibitor Cocktail 1X (Roche)) using a FastPrep® 24 instrument (MP Biomedical) for 45 s at speed 6.5, for use as fresh substrate (without a congelation step). Infected brain tissues were prepared similarly and stored at −80 °C, and used as seed for PMCA reaction.
Ten percent (wt/vol) normal brain homogenates (NBH) were prepared in conversion buffer (PBS with 150 mM NaCl, 1% Triton X-100, 0.005% EDTA pH 8, and Complete Protease Inhibitor Cocktail 1X (Roche)) using a FastPrep® 24 instrument (MP Biomedical) for 45 s at speed 6.5, for use as fresh substrate (without a congelation step). Infected brain tissues were prepared similarly and stored at −80 °C, and used as seed for PMCA reaction.
3
Protein Extraction from Tissues
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Proteins were extracted from kidneys, testes, brain, skeletal muscle and heart. Therefore the frozen tissues were disrupted and homogenized by a mortar and pestle in liquid nitrogen. 10-45 mg of the frozen tissue powder was transferred to RIPA lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 µM EGTA, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholic acid, 2% complete protease inhibitor cocktail 1x (Roche, Basel, Switzerland)) and stepwise sonicated (Ultrasonic converters UW 2070, Berlin, Germany). After centrifugation at 20,000 x g for 30 minutes at 4°C, the supernatants containing the extracted proteins were stored at -80°C.
Protein determination was conducted by using the Bicinchoninic Acid Kit (Sigma-Aldrich, Munich, Germany) and a microplate reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland).
Protein determination was conducted by using the Bicinchoninic Acid Kit (Sigma-Aldrich, Munich, Germany) and a microplate reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland).
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