For protein expression analyses yeast were harvested during the exponential phase of growth at an OD600 of 0.6, separated by filtration (see below) and cryogenically grinded in a SPEX Freezer Mill. The samples were resuspended in Y‐PER yeast protein extraction reagent (Thermo Fisher) and cellular debris removed by centrifugation. Each sample was chemically labeled separately with one of up to eight distinct isobaric iTRAQ reagents (Ross et al., 2004 ). Peak lists were extracted using the MASCOT Distiller software (MatrixScience). False discovery rates (FDR) were estimated by searching equivalent reversed or randomized sequence databases, using a cutoff value of 1% FDR. Protein‐level expression ratios were calculated using intensity‐weighted averages from matching peptides after outlier removal. This experiment was performed in duplicate. Comparing the two sample replicates, we fitted a Cauchy distribution using the EasyFit statistical package (MathWave, Inc.). Peptide p‐values were calculated using a two‐tailed t‐test.
Y per yeast protein extraction reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Y-PER Yeast Protein Extraction Reagent is a solution designed to facilitate the extraction of proteins from yeast cells. It is a ready-to-use reagent that does not require additional components for the extraction process.
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Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
M iggk bp hrp second antibody, by Santa Cruz Biotechnology (3 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Mascot distiller software, by Matrix Science (1 mentions)
Glutathione sepharose column, by GE Healthcare (1 mentions)
Isopropyl β d thiogalactopyranoside, by Fujifilm (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Pierce 1 step ultra tmb blotting solution, by Thermo Fisher Scientific (1 mentions)
Rnaeasy mini kit, by Qiagen (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Hybond, by Cytiva (1 mentions)
Amicon ultra 4 centrifugal filter unit, by Merck Group (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Bradford reagent, by Aidlab (1 mentions)
21 protocols using y per yeast protein extraction reagent
1
Quantitative Proteomic Analysis of Yeast Samples
2
Recombinant ING1 Protein Purification
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We transformed ECOS competent Escherichia coli JM-109 cells (Nippon Gene) with the recombinant plasmid pGEX-4 T-1-ING1 and cultured them for 3 h in 200-ml Luria broth containing 0.1 mM isopropyl β-D-thiogalactopyranoside (FUJIFILM Wako Pure Chemical, Osaka, Japan). We then harvested the cells, washed them with phosphate-buffered saline, and lysed them by sonication in Y-PER Yeast Protein Extraction Reagent (Thermo Fisher Scientific). We centrifuged the lysates at 15,000 g for 10 min at 4 °C and purified the GST-fused ING1 proteins in supernatants using affinity chromatography with Glutathione-Sepharose columns (GE Healthcare Life Sciences) according to the manufacturer's instructions. We finally concentrated the purified proteins with an Amicon Ultra-15 Centrifugal Filter Device (Merck KGaA, Millipore, Darmstadt, Germany) as described [12 (link), 13 (link)].
3
Colorimetric Assay for CaLB Lipase Activity
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The enzymatic activity of Candida antarctica lipase B (CaLB) was determined according to the colorimetric assay using p-nitrophenyl butyrate (pNPB) as a substrate (Krainer et al., 2012 (link)). 20 μL of the enzyme sample (culture supernatant) was transferred into a 96-well microtiter plate, and 180 μL of a freshly prepared pNPB working solution (100 μL pNPB stock solution in 10 mL of 300 mM Tris/HCl buffer, pH 8.0; pNPB stock solution was prepared by mixing 42 μL of pNPB (Sigma Aldrich) with 458 μL of DMSO and stored at −20°C) was added. The kinetics of the reaction were measured immediately at 405 nm for 6 min at 25°C. One activity unit (U) of CaLB was defined as the amount of CaLB that was needed to produce 1 μmol p-nitrophenolate mL−1 min−1.
For the measurement of the intracellular CaLB, centrifuged cells from a 0.5 mL sample were washed with PBS and stored at −20°C. The cell pellet was gently resuspended in Y-PER™ Yeast Protein Extraction Reagent (Thermo Scientific, Rockford, USA) in ratio 2.5 μL Y-PER per 1 mg WCW. The suspension was then incubated in the Eppendorf agitator at room temperature for 30 min and 600 rpm. Then, the sample was centrifuged (14,000 rpm, 10 min) and the supernatant was used for the CaLB activity assay.
For the measurement of the intracellular CaLB, centrifuged cells from a 0.5 mL sample were washed with PBS and stored at −20°C. The cell pellet was gently resuspended in Y-PER™ Yeast Protein Extraction Reagent (Thermo Scientific, Rockford, USA) in ratio 2.5 μL Y-PER per 1 mg WCW. The suspension was then incubated in the Eppendorf agitator at room temperature for 30 min and 600 rpm. Then, the sample was centrifuged (14,000 rpm, 10 min) and the supernatant was used for the CaLB activity assay.
4
Yeast Protein Extraction Protocol
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Yeast cells pellets were resuspended in an appropriate amount of Y-PER Yeast Protein Extraction Reagent (Thermo Scientific) added of 100X Halt Protease Inhibitor Cocktail (Thermo Scientific). The amount of Y-PER Yeast Protein Extraction Reagent was calculated according to the weight of pellet as indicated the manufacturer’s instructions. The mixtures ware agitated at room temperature for 20 minutes and then, centrifuged at 14,000g for 10 minutes at 4°C. The supernatant that contains proteins was reserved and stored at -80°C. Protein extracts were quantified using Pierce BCA Protein Assay Kit (Thermo Scientific).
5
Yeast Protein Extraction and Assay
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Example 4
150 μL of Y-PER yeast protein extraction reagent (ThermoFisher Scientific, USA: Catalogue #78990) was added to the cell paste in each well as described above. The cells were lysed at room temperature for 1.5 hrs. lysis method was used with shaking on a bench top shaker. The plate was then centrifuged for 10 min at 4000 rpm and 4° C. The clear supernatants were used to perform biochemical assays to determine activity.
6
Yeast Protein Immunoblotting Protocol
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Cell extracts were extracted in Y-PER
Yeast Protein Extraction Reagent (ThermoFisher) with the addition
of an ethylenediaminetetraacetic acid-free protease inhibitor (Roche).
Cell lysates (25 μL) were resolved using a 15% SDS-PAGE gel.
The resolved proteins were electro-transferred to polyvinylidene difluoride
membranes (Bio-Rad), which were then probed with HRP-conjugated anti-6×
His tag antibody (Rabbit polyclonal) (abcam) in 1:4000 dilution. The
results were visualized using a Pierce 1-step ultra TMB blotting solution
(ThermoFisher).
Yeast Protein Extraction Reagent (ThermoFisher) with the addition
of an ethylenediaminetetraacetic acid-free protease inhibitor (Roche).
Cell lysates (25 μL) were resolved using a 15% SDS-PAGE gel.
The resolved proteins were electro-transferred to polyvinylidene difluoride
membranes (Bio-Rad), which were then probed with HRP-conjugated anti-6×
His tag antibody (Rabbit polyclonal) (abcam) in 1:4000 dilution. The
results were visualized using a Pierce 1-step ultra TMB blotting solution
(ThermoFisher).
7
Hsu1-GFP Protein Purification Protocol
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The strain was streaked from the GFP library and the coding region was verified via specific primers [20 (link)] (S3 Table ). To maximize the recovery of Hsu1-GFP protein, the strain was cultured for 5 days in 150 ml minimal media supplemented with leucine, uracil, and 0.4 mM NaHS at 25°C. Following this, cells were pelleted by centrifugation at 3,220g for 5 min at 4°C and resuspended in an appropriate volume of Y-PER Yeast Protein Extraction Reagent (Thermo Fisher) containing 1× Protease Inhibitor cocktail (P8215, Sigma). Purified protein extract was obtained following the manufacturer’s protocol, to which 250 μl of GFP-clamp resin (in-house, Crick-Structural Biology Scientific Technology Platform) was added to the lysis solution and incubated at 4°C for 2 h with rotation.
8
Yeast Protein and RNA Extraction Protocol
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Example 7
Three colonies were picked per each variant. Transgenic yeast cells were grown overnight in YPD broth containing G-418 at 30° C. with shaking at 255 rpm. The following morning, cultures were diluted to an O.D. of about 0.5 at 600 nm. Three to four hours later when the O.D. of the cultures reached about 1.0 at 600 nm, the cells were harvested. Two ml for each variant was used and total protein and total RNA were isolated.
Protein was isolated using Y-PER Yeast Protein Extraction Reagent (Thermo Scientific, Rockford, Ill.). Total RNA was isolated using RNA Easy Mini Kit (QIAGEN). To generate spheroplasts, yeast cells were resuspended in 2 ml of Buffer Y containing zymolase and incubated 30 min at 30° C. with gentle shaking at 75 rpm. Buffer Y was prepared by adding 50× (10 mg/ml) zymolase-100T (from Arthrobacter luteus; Seikagaku Biobusiness Corporation, Japan) and 0.1% β-mercaptoehtanol to 1M sorbitol and 0.1 EDTA, pH 7.4 solution. To eliminate DNA contamination, RNA samples were digested with RNAse-Free DNase Set (QIAGEN) according to the manufacturer's protocol.
Three protein and three RNA samples were analyzed by ELISA and quantitative PCR for each variant.
9
Yeast Protein Extraction Protocol
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To exclude significant loss in cell mass, protein was extracted using Y-PER Yeast Protein Extraction Reagent (Thermo Scientific). Yeast numbers, germination conditions and timing were the same as those for the RNA extraction and XTT experiments. After the incubation periods, cells were pelleted at 3000 x g for 5 minutes at 4°C. The pellets were suspended in 250 μL Y-PER reagent and vortexed gently at room temperature for 20 minutes. The mixture was centrifuged at 14,000 x g for 10 minutes saving the supernatant containing the soluble protein. The protein concentration was measured with the Qubit 2.0 fluorometer using the Qubit Protein Assay Kit (Life Technologies).
10
Protein Extraction and SDS-PAGE Analysis
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Cells were pelleted by centrifugation at 4000 rpm for 5 min. The cells were processed immediately after centrifugation or pellets were frozen at – 80 °C. The cells were then resuspended such that 100 μl of Y-PER™ Yeast Protein Extraction Reagent (78990) (Thermo Fisher Scientific Inc., Altrincham, UK ) were used to resuspend 50 mg of dry cell pellet. SDS PAGE gel electrophoresis was carried out using Bio-Rad Mini Protean II kits (Bio-Rad Laboratories Ltd., Watford, UK). The western blotting technique is based on the technique described by W. N. Burnette [69 (link)].
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