Y-PER™ Yeast Protein Extraction Reagent

Protocol tips

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	- Cells are resuspended in Yeast Protein Extraction Reagent and the isolation is performed according to the manufacturer's protocol.

Protocol tips
- Cells are resuspended in Yeast Protein Extraction Reagent and the isolation is performed according to the manufacturer's protocol.

Publication protocol

Enzyme assays were carried out as described previously, with modifications [23]. Briefly, a primary inoculum culture was prepared in YPD medium, with zeocin (100 μg/mL), and maintained at 30 °C and 180 rpm overnight. Cells were harvested, re-inoculated in a new flask, and grown in a shaker at 30 °C until the exponential phase. After centrifugation, cells were resuspended in Yeast Protein Extraction Reagent (Y-Per, Thermo Scientific, Rockford, IL, USA) for 10 min. The reaction was assembled with 10 µL cellular extract, 8 µL NADH, 800 µL 50 mM phosphate buffer (pH 7), and ultra-pure water for a 1 mL final volume. After 150 s, 40 µL of pyruvate was added and the reaction was completed in 300 s. A unit of enzyme activity was defined as the amount of enzyme necessary to oxidize 1 μmol of NADH per minute. Protein concentration of cell extracts was determined using the Coomassie protein assay reagent (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. BSA in known concentrations was used to construct the standard curve. Enzyme assays were carried out in three biological replicates.

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Y-PER™ Yeast Protein Extraction Reagent below.

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