The seedlings were transferred into a liquid N2 chilled 35 ml ball mill and disrupted in a reciprocal mixer mill [30 Hz, 45 s, repeated three times (Retsch USA)] under liquid nitrogen. Ground tissue was gently resuspended in 1 ml (approximately 1 packed tissue volume) of SII buffer (100 mM sodium phosphate, pH 8.0, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Triton X-100, 1 mM PMSF, 1x protease inhibitor cocktail [Roche], 1x phosphatase inhibitors II & III [Sigma], and 50 μM Mg-132 [Peptides International]) and sonicated twice at 40% power, 1 s on/off cycles for 20 s total on ice (Fisher Scientific model FB505, with microtip probe). Extracts were clarified by centrifugation twice at 4 °C for 10 min at ≥20,000g, then subjected to cold acetone precipitation to remove buffer contaminants and lipophilic metabolite contaminants. Protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific). Protein samples were reduced with 10 mM tris(2-carboxyethyl)phosphine and alkylated with 25 mM iodoacetamide before trypsin digestion in 1/40 enzyme/protein ratio at 37 °C overnight. Finally, samples were desalted with a C18 column before dry down.
Phosphatase inhibitors 2 3
Manufactured by Merck Group
Phosphatase inhibitors II & III are a series of chemical compounds used in laboratory research to inhibit the activity of phosphatases, which are enzymes that remove phosphate groups from other molecules. These inhibitors are commonly used in various biochemical and cell-based assays to study signal transduction pathways, protein function, and other cellular processes involving phosphorylation.
Automatically generated - may contain errors
2 protocols using phosphatase inhibitors 2 3
1
Protein Extraction and Purification Protocol
2
Protein Extraction and Preparation
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The seedlings were transferred into a liquid N2 chilled 35ml ball mill and disrupted in a reciprocal mixer mill [30 Hz, 45 seconds, repeated three times (Retsch USA)] under liquid nitrogen. Ground tissue was gently resuspended in 1 mL (approximately 1 packed tissue volume) of SII buffer (100 mM sodium phosphate, pH 8.0, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% Triton X-100, 1 mM PMSF, 1x protease inhibitor cocktail [Roche], 1x Phosphatase Inhibitors II & III [Sigma], and 50 µM Mg-132 [Peptides International]) and sonicated twice at 40% power, 1 second on/off cycles for 20 s total on ice (Fisher Scientific model FB505, with microtip probe). Extracts were clarified by centrifugation twice at 4˚C for 10 min at ≥20,000xg. Protein concentrations were determined by BCA protein assay (Thermo-Fisher Scientific, Rockford, IL). Protein samples were reduced with 10 mM TCEP and alkylated with 25 mM iodoacetamide before trypsin digestion in 1/40 enzyme/protein ratio at 37°C overnight.
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