Cells were lysed in a buffer containing 20 mM HEPES, 150 mM NaCl, 10% (v/v) glycerol, 1 mM EGTA, 1.5 mM MgCl2, 1% (v/v) Triton X-100, pH to 7.4, protease inhibitor cocktail (Sigma-Aldrich), and protein phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich), and incubated in an ice/water slurry for 20 min, followed by 2 freezethaw cycles (-80°C/37°C, ~ 1 min each). Supernatant was collected after ultracentrifugation at 100,000 g, 4°C, for 30 min. Protein concentration was determined using BCA assay (Pierce, Thermo Fisher Scientific). Endoglycosidase (Endo) H (New England Biolabs) digestion was performed based on the manufacturer’s instructions. Briefly, 20–40 μg bulk protein was assembled in 15.3 μl reaction volume; 1.7 μl denaturing buffer was added and samples were boiled for 10 min at 100°C. Then 2 μl of G5 buffer and 1 μl of Endo H or 1 μl H2O were added to the denatured reaction and incubated for 2 hours at 37°C.
Protein phosphatase inhibitor cocktail 1 and 2
Manufactured by Merck Group
Protein phosphatase inhibitor cocktails 1 and 2 are laboratory reagents used to inhibit the activity of protein phosphatases. Cocktail 1 contains a mixture of small molecule inhibitors, while cocktail 2 contains a different set of inhibitors. These cocktails are commonly used in biochemical and cell-based experiments to study the role of protein phosphorylation in various cellular processes.
Automatically generated - may contain errors
2 protocols using protein phosphatase inhibitor cocktail 1 and 2
1
Protein Glycosylation Analysis Using Endo H
2
Phosphoproteome Enrichment and Analysis
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Cells were washed and harvested with ice-cold PBS. The proteins were extracted with phase-transfer surfactant (34 (link)) using a lysis buffer (12 mM sodium deoxycholate [Fujifilm Wako], 12 mM sodium N-lauroylsarcosinate [Fujifilm Wako], 100 mM Tris-HCl [pH 9.0], containing protein phosphatase inhibitor cocktail 1 and 2 [Sigma-Aldrich], and protease inhibitors [Sigma-Aldrich]). Protein amount was determined with a BCA protein assay kit, and the proteins were reduced with 10 mM DTT for 30 min and then alkylated with 50 mM iodoacetamide for 30 min in the dark. After reduction and alkylation, proteins were digested with Lys-C (w/w 1:100) for 3 h, followed by trypsin digestion (w/w 1:100) overnight at 37 °C. Then, the peptides were desalted using SDB-XC StageTip.
Phosphopeptides were enriched from 100 μg of tryptic peptides by means of TiO2-based hydroxy-acid-modified metal oxide chromatography (HAMMOC) (35 (link)) and eluted with 0.5% piperidine. Phosphopeptides were labeled with tandem mass tag (TMT) (Thermo Fisher Scientific), desalted using SDB-XC StageTips, fractionated at basic pH (33 (link)), and suspended in the loading buffer (0.5% TFA and 4% ACN) for subsequent LC/MS/MS analyses.
Phosphopeptides were enriched from 100 μg of tryptic peptides by means of TiO2-based hydroxy-acid-modified metal oxide chromatography (HAMMOC) (35 (link)) and eluted with 0.5% piperidine. Phosphopeptides were labeled with tandem mass tag (TMT) (Thermo Fisher Scientific), desalted using SDB-XC StageTips, fractionated at basic pH (33 (link)), and suspended in the loading buffer (0.5% TFA and 4% ACN) for subsequent LC/MS/MS analyses.
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