The composition of the culture media was carefully adjusted to minimize background fluorescence (FL), provide sufficient amounts of carbon (C) and nitrogen (N) to avoid starvation, and maintain the pH stably throughout the culturing time. The medium was composed as follows [g L−1]: yeast nitrogen base without amino acids and ammonium sulfate, 5.1 (Sigma-Aldrich); ammonium sulfate or casamino acid hydrolysate, 15, (POCH); glucose or glycerol, 35 (POCH); buffered with 0.1 or 0.2 M maleic acid (POCH). Precultures were developed in media composed of a yeast nitrogen base, 5.1 (Sigma-Aldrich); ammonium sulfate, 15, (POCH); glucose, 25 (POCH); buffered with 0.2 M maleic acid at pH 5.0. All the media were filter-sterilized with 0.22-μm filters (Merck- Millipore, Darmstadt, Germany).
Ammonium sulfate
Manufactured by Merck Group
Sourced in United States, Germany, Belgium, France, Denmark, Netherlands, Brazil
Ammonium sulfate is a chemical compound with the formula (NH4)2SO4. It is commonly used as a laboratory reagent and in various industrial applications. Ammonium sulfate is a crystalline, water-soluble solid that serves as a source of both nitrogen and sulfur for various chemical processes and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Merck Group (25 mentions)
Sodium hydroxide (naoh), by Merck Group (23 mentions)
Hydrochloric acid (hcl), by Merck Group (13 mentions)
Bovine serum albumin (bsa), by Merck Group (12 mentions)
Sulfuric acid, by Merck Group (11 mentions)
Ethanol, by Merck Group (10 mentions)
Glycerol, by Merck Group (9 mentions)
Glycine, by Merck Group (9 mentions)
Tween 20, by Merck Group (9 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (9 mentions)
D glucose, by Merck Group (9 mentions)
Acetic acid, by Merck Group (9 mentions)
Quercetin, by Merck Group (7 mentions)
Triton x 100, by Merck Group (7 mentions)
Cholesterol, by Merck Group (7 mentions)
Sodium acetate, by Merck Group (7 mentions)
Sodium carbonate, by Merck Group (7 mentions)
Methanol, by Merck Group (7 mentions)
Magnesium sulfate, by Merck Group (6 mentions)
234 protocols using ammonium sulfate
1
Optimized Culture Media for Fluorescence Imaging
2
Yeast Auxotroph Strain Engineering
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The bacterial strains, plasmids, and
primers used in this study are
listed in the Supporting Information,Table S1 . Escherichia coli NEB5α high efficiency competent
cells were obtained from NEB (New England Biolabs Ipswich, MA). The
auxotrophic Y. lipolytica Po1g (Leu−) was
purchased from Yeastern Biotech Company (Taipei, Taiwan). Y. lipolytica XP1 harboring the plasmid pYaliA1-vioDCBAE
was stored in our lab.39 (link) YPD medium contains
20 g/L glucose (Sigma-Aldrich), 10 g/L yeast extract (Sigma-Aldrich),
and 20 g/L peptone (Sigma-Aldrich). YNB medium (C/N = 60, 80, 100,
120) contains 1.7 g/L yeast nitrogen base (without amino acids and
ammonium sulfate) (Difco), 1.1 g/L ammonium sulfate (Sigma-Aldrich),
0.69 g/L CSM-Leu (Sunrise Science Products, Inc.), and 30, 40, 50,
60 g/L glucose, respectively. YNB medium (pH 6.0, 6.5, 7.0, 7.5) contains
1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate)
(Difco), 1.1 g/L ammonium sulfate (Sigma-Aldrich), 0.69 g/L CSM-Leu
(Sunrise Science Products, Inc.), 30 g/L glucose, and was adjusted
to pH 6.0, 6.5, 7.0, 7.5, respectively, through Na2HPO4 and NaH2PO4. YNB medium with CaCO3 was made with YNB media supplemented with 10 g/L CaCO3. Selective YNB plates were made with YNB media supplemented
with 20 g/L Bacto agar (Difco).
primers used in this study are
listed in the Supporting Information,
cells were obtained from NEB (New England Biolabs Ipswich, MA). The
auxotrophic Y. lipolytica Po1g (Leu−) was
purchased from Yeastern Biotech Company (Taipei, Taiwan). Y. lipolytica XP1 harboring the plasmid pYaliA1-vioDCBAE
was stored in our lab.39 (link) YPD medium contains
20 g/L glucose (Sigma-Aldrich), 10 g/L yeast extract (Sigma-Aldrich),
and 20 g/L peptone (Sigma-Aldrich). YNB medium (C/N = 60, 80, 100,
120) contains 1.7 g/L yeast nitrogen base (without amino acids and
ammonium sulfate) (Difco), 1.1 g/L ammonium sulfate (Sigma-Aldrich),
0.69 g/L CSM-Leu (Sunrise Science Products, Inc.), and 30, 40, 50,
60 g/L glucose, respectively. YNB medium (pH 6.0, 6.5, 7.0, 7.5) contains
1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate)
(Difco), 1.1 g/L ammonium sulfate (Sigma-Aldrich), 0.69 g/L CSM-Leu
(Sunrise Science Products, Inc.), 30 g/L glucose, and was adjusted
to pH 6.0, 6.5, 7.0, 7.5, respectively, through Na2HPO4 and NaH2PO4. YNB medium with CaCO3 was made with YNB media supplemented with 10 g/L CaCO3. Selective YNB plates were made with YNB media supplemented
with 20 g/L Bacto agar (Difco).
3
Proteinase K and Ammonium Sulfate Purification
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For the proteinase K treatment, LPS-free supernatant was digested with proteinase K (0.2 mg/ml; Sigma) at 55°C overnight, following incubation in boiling water for 30 min. The supernatant was then centrifuged at 13,000 rpm for 1 min, and the supernatant was collected and used to vaccinate the mice. For the ammonium sulfate precipitation step, ammonium sulfate (Sigma) at 20% (final concentration) was added to the supernatant and incubated at 4°C with shaking for 1 h. The mixture was centrifuged at 13,000 rpm for 15 min, and the supernatant and pellet were collected. ammonium sulfate at 50% (final concentration) was added to the supernatant fraction from the 20% precipitation, and the same procedure described above was performed. The pellets of the 20% and 50% fractions and the supernatant of the 50% fraction were dialyzed in PBS and used to vaccinate the mice.
4
Yeast Strain Engineering for GRX1 Study
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The yeast strain GRX1-GFP-HIS3MX6 was purchased from Thermo Ficher Scientific. All the primers used in this study are listed in Supplementary Table 1 . To create the GRX1-GFP-tdTomato-kanR strain, a PCR fragment containing tdTomato-kanR and homologies to GFP-HIS3MX6 was amplified with primers C1 and C2 from pfa6a-tdTomato-kanR (constructed in our lab) and transformed to the GRX1-GFP-HIS3MX6 strain.
All the strains were grown in liquid YPD medium containing 20 g/L glucose (Sigma), 10 g/L peptone (Euromedex) and 10 g/L yeast extraction (Euromedex). When needed, 20 g/L agar (Euromedex) was added to make solid plates. YNB-URA− plates [20 g/L glucose (Sigma), 20 g/L agar (Euromedex), 1.71 g/L yeast nitrogen base without amino acids and nitrogen (Euromedex), 5 g/L ammonium sulfate (Sigma), and 0.77 g/L CSM-URA− (Euromedex)] or 5-FOA plate [20 g/L glucose (Sigma), 20 g/L agar (Euromedex), 1.71 g/L yeast nitrogen base without amino acids and nitrogen (Euromedex), 5 g/L ammonium sulfate (Sigma), 0.79 g/L CSM-URA− (Euromedex), and 1 g/L 5-FOA (Euromedex)] were used to select transformants.
All the strains were grown in liquid YPD medium containing 20 g/L glucose (Sigma), 10 g/L peptone (Euromedex) and 10 g/L yeast extraction (Euromedex). When needed, 20 g/L agar (Euromedex) was added to make solid plates. YNB-URA− plates [20 g/L glucose (Sigma), 20 g/L agar (Euromedex), 1.71 g/L yeast nitrogen base without amino acids and nitrogen (Euromedex), 5 g/L ammonium sulfate (Sigma), and 0.77 g/L CSM-URA− (Euromedex)] or 5-FOA plate [20 g/L glucose (Sigma), 20 g/L agar (Euromedex), 1.71 g/L yeast nitrogen base without amino acids and nitrogen (Euromedex), 5 g/L ammonium sulfate (Sigma), 0.79 g/L CSM-URA− (Euromedex), and 1 g/L 5-FOA (Euromedex)] were used to select transformants.
5
Yeast Genetic Manipulation and Protein Expression
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All yeast strains used in this study are detailed in Table S1. Yeast genetic manipulations were conducted using classical yeast knock-in/out protocols. For ectopic protein expression, genes were cloned into pBP73-C or -G vectors encoding either the GPD promoter or CPY promoter. All plasmids used in this study are detailed in Table S2. Yeast transformations were performed using the lithium acetate method. We used Ds-Red HDEL to label the ER. Strains were selected using antibiotics or dropout selection media. All chemicals used to make the yeast media were purchased from Sigma-Aldrich (succinic acid, sodium hydroxide, ammonium sulfate, yeast nitrogen base without amino acids, or ammonium sulfate). Yeast media was supplemented with a final concentration of 2% dextrose unless otherwise indicated. oleate (Sigma-Aldrich; O1008) and POA (Sigma-Aldrich; P9417) were added to the culture media as indicated (0.2% oleate is equivalent to 6.32 mM, and 0.2% POA is equivalent to 7.04 mM).
6
Crystallization of hSpt2C-H3/H4 Tetramer
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The reconstituted hSpt2C–H3/H4 tetramer complex was collected by pooling Hiload 16/60 Superdex 200 (GE Healthcare) gel filtration chromatographic peak fractions to a concentration of 5 mg/mL for crystallization trials. Crystals of the hSpt2C–H3/H4 tetramer complex were grown from a solution containing 0.02 M NaCl (Fluke), 0.2 M HEPES 7.5 (Sigma-Aldrich), and 1.5 M ammonium sulfate (Sigma-Aldrich) at 20°C using the hanging drop vapor diffusion method. For data collection, crystals were flash-frozen (100 K) in the above reservoir solution supplemented with 2 M Li-sulfate (Sigma-Aldrich). A diffraction data set was collected to 3.3 Å resolution at wavelength 0.9798 Å on NE-CAT beam line 24ID-E at the Advanced Photon Source, Argonne National Laboratory. Crystals of the hSpt2C(Se-Met, I615M)–H3/H4 tetramer complex were grown under conditions similar to that of the native complex from a solution containing 0.02 M NaCl (Fluke), 0.2 M HEPES 7.5 (Sigma-Aldrich), and 1.6 M ammonium sulfate (Sigma-Aldrich) at 20°C using the hanging drop vapor diffusion method. For data collection, crystals were flash-frozen (100 K) in the above reservoir solution supplemented with 20% glycerol (Sigma-Aldrich). A diffraction data set was collected to 4.6 Å resolution at wavelength 0.9792 Å on NE-CAT beam line 24ID-E at the Advanced Photon Source, Argonne National Laboratory.
7
Proteomics Workflow for T3 Peptide Quantification
8
Yeast Transformation Using Alkali-Cation Kit
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The S. cerevisiae yeast strain, INVSc1 (genotype: MATa his3Δ1 leu2 trp1-289 ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-52), was obtained from Invitrogen. Transformation of the INVSc1 yeast was performed using an Alkali-Cation Yeast Transformation kit (MP Biomedicals) according to the manufacturer's instructions with 2 mL yeast preculture grown overnight in 100 mL YPD medium to an absorbance at 660 nm equivalent to an optical density of 1.6 (DU-640 Spectrometer; Beckman Coulter, Fullerton, CA). Transformants were selected by plating onto 2% glucose complete minimal (CM) medium minus uracil plates (1.4 g yeast synthetic dropout medium supplement without histidine, tryptophan, leucine, or uracil [Sigma-Aldrich]; 6.7 g yeast nitrogen base with ammonium sulfate [Sigma-Aldrich]; 20 g D-glucose [Fisher Scientific, Fair Lawn, NJ]; 76 mg each of histidine, tryptophan, and leucine; and 20 g Bacto Agar [Sigma-Aldrich] in a final volume of 1 L Milli-Q water) or onto 2% xylose CM medium plates (1.4 g yeast synthetic dropout medium supplement without histidine, tryptophan, leucine, or uracil; 6.7 g yeast nitrogen base with ammonium sulfate; 20 g D-xylose; 76 mg each of histidine, tryptophan, leucine, and uracil; and 20 g Bacto Agar in a final volume of 1 L Milli-Q water).
9
Hydrophobic Interaction Chromatography of Antibodies
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For hydrophobic interaction chromatography (HIC-HPLC), the samples were adjusted to an antibody concentration of 0.5 mg/mL and 0.5 M ammonium sulfate (Merck KgaA, Germany). Then, 80 µL (40 µg) was injected into a TSKgel Butyl NPR (Tosoh, Tokyo, Japan) column, and separation was performed using a full linear gradient from A (1.2 M ammonium sulfate (Merck KgaA, Germany), 1× PBS pH 7.2 (Invitrogen, Carlsbad, CA, USA)) to B (50% (v/v) methanol (VWR, Atlanta, GA, USA), 0.1× PBS, pH 7.2) within 15 min at a 0.5 mL/min flow rate, recording the absorption at 280 nm wavelength.
10
Synthesis and Characterization of Bis-2,2-1H-imidazole
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Bis-2,2-1H-imidazole was synthesized for this study using the procedure described in ref. 13 and 14. Its purity was determined by 1 H-NMR to be Z99%. All other compounds were purchased from Aldrich: imidazole, 99%; imidazole carboxaldehyde, 99%; glyoxal, aqueous solution 40 wt% (8.8 M); ammonium sulfate, 99%; D 2 O, 99.9 atom% D; acetonitrile 99.8%; trimethylsilyl propanoic acid (TMSP) 98 atom% D.
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