Y-PER™ Yeast Protein Extraction Reagent

Protocol tips

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	- Cell pellets are resuspended in Y-PER yeast protein extraction reagent, which contained EDTA-free pierce protease inhibitors, and were incubated with agitation at room temperature for 20 min. Lysed cell samples are then centrifuged at 17,000 × g for 10 min, and the soluble fractions are collected for enzyme assays.

Protocol tips
- Cell pellets are resuspended in Y-PER yeast protein extraction reagent, which contained EDTA-free pierce protease inhibitors, and were incubated with agitation at room temperature for 20 min. Lysed cell samples are then centrifuged at 17,000 × g for 10 min, and the soluble fractions are collected for enzyme assays.

Publication protocol

To prepare samples for cell-free enzyme assays, 1 × 107 cells in late exponential growth were collected and centrifuged at 4,700 × g for 3 min. Cell pellets were resuspended in Y-PER yeast protein extraction reagent (catalog no.78990; Thermo Scientific, IL, USA), which contained EDTA-free pierce protease inhibitors (catalog no. 88266; Thermos Scientific, IL, USA), and were incubated with agitation at room temperature for 20 min. Lysed cell samples were then centrifuged at 17,000 × g for 10 min, and the soluble fractions were collected for enzyme assays. Unless specified, all the enzyme assay reactions were conducted in 384-well plates with a 50-μl working reaction volume at 28°C. Kinetics measurement was conducted with a BioTek Synergy HT microplate reader, and data were processed using Gen5 software (BioTek Instruments, Inc., VT, USA). Protein concentrations were measured by the Bradford assay.

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