Samples were incubated with 0.04 µg dithiothreitol (DTT, Sigma-Aldrich, St. Louis, MO, USA) per µg protein at 56 °C for 30 min. After incubation with 0.2 µg iodoacetamide (IAA, Sigma-Aldrich, St. Louis, MO, USA) per µg protein for 30 min at RT in the dark, the reaction was quenched with 0.2 µg DTT per µg protein. Sequencing-grade trypsin (1 µg per 75 µg protein, Promega, Fitchburg, WI, USA) was added and samples were incubated for 4 h at 37 °C. After a further aliquot of trypsin (1 µg per 75 µg protein) was added, the samples were incubated at 37 °C overnight. The digestion was stopped by acidifying the samples to a pH of 2.5 by adding trifluoroacetic acid (Thermo Fisher Scientific, Waltham, MA, USA).
Dithiothreitol (dtt)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, Sao Tome and Principe, France, China, Switzerland, Macao, Spain, Australia, Canada, Belgium, Sweden, Brazil, Austria, Israel, Japan, New Zealand, Poland, Bulgaria
Dithiothreitol (DTT) is a reducing agent commonly used in biochemical and molecular biology applications. It is a small, water-soluble compound that helps maintain reducing conditions and prevent oxidation of sulfhydryl groups in proteins and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Iodoacetamide, by Merck Group (502 mentions)
Ammonium bicarbonate, by Merck Group (194 mentions)
Trypsin, by Promega (178 mentions)
Urea, by Merck Group (177 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (163 mentions)
Formic acid, by Merck Group (151 mentions)
Bovine serum albumin (bsa), by Merck Group (123 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (104 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (97 mentions)
Sodium chloride (nacl), by Merck Group (95 mentions)
Triton x 100, by Merck Group (92 mentions)
Trypsin, by Merck Group (84 mentions)
Trifluoroacetic acid, by Merck Group (83 mentions)
Hepes, by Merck Group (76 mentions)
Formic acid, by Thermo Fisher Scientific (75 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (72 mentions)
Acetonitrile, by Merck Group (71 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (69 mentions)
Sequencing grade modified trypsin, by Promega (66 mentions)
Protease inhibitor cocktail, by Merck Group (65 mentions)
2 532 protocols using dithiothreitol (dtt)
1
Trypsin Digestion of Protein Samples
2
Membrane Protein Extraction and Glycan Release
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A sample of membrane protein extraction from cells (100–200 μg of protein) was added at 1/10 of the sample volume of 1 M ammonium bicarbonate (Sigma-Aldrich) and 5 μL of 0.2 M dithiothreitol (DTT, Sigma-Aldrich). The sample mixture was reduced by DTT at 60 °C for 30 min followed by alkylation with 5 μL of 0.5 M iodoacetamide (IAA, Sigma-Aldrich) by incubation in the dark at room temperature for 30 min. Then, the sample mixture was incubated at room temperature with 1.25 μL of 0.2 M DTT to remove excess IAA. The sample mixture was then treated with 5 μL of 200 ng/μL trypsin (Sigma-Aldrich) at 37 °C for at least 5 h with shaking (1000 rpm), followed by heat inactivation of the enzyme at 100 °C for 5 min. N-Glycans were enzymatically released from trypsin-digested glycopeptides by incubation with 1 μL Rapid PNGase F (New England Biolabs, Ipswich, MA, USA) at 37 °C for at least 16 h with shaking (1000 rpm).
3
Fabrication of AM1241-Loaded PEG-DTT Hydrogels
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PEG-DTT hydrogels were prepared by the Michael addition reaction using poly(ethylene glycol) diacrylate (PEGDA) (molecular weight: 700, Sigma-Aldrich, United States) and DTT (Sigma-Aldrich, United States) over the sodium tetraborate catalyst. AM1241 (0, 1, 2.5, 5, 10, 20, 40, 80, 120, and 200 µM) (Sigma-Aldrich, United States) was then immediately added to this solution and mixed to prepare a finished AM1241-loaded PEG-DTT hydrogel. First, 100 mg of PEGDA (0.14 mM) and 22 mg of DTT (0.14 mM) were dissolved in 0.5 ml of water. Then, 0.5 ml of 0.1 mol/L borax (Sigma-Aldrich, United States) solution was added to the mixture and stirred vigorously for 10 s. Keep the solution at room temperature (25°C) for a while and check the gelation time by tube inversion.
4
Protein Extraction and Precipitation Protocol
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The protocol reported by Niu et al. [12 (link)] was used for protein extraction and precipitation with some modifications. The supernatants were precipitated for 6 hours by adding 5 ml of ice-cold trichloroacetic acid (TCA)/acetone buffer (10% TCA (Merck) and 10 mM dithiothreitol (DTT)(Sigma-Aldrich) in acetone (Fisher Scientific)) to 1 ml of the sample. Then, the sample was centrifuged at 15,000 g for 10 min at 4°C, and the supernatant was discarded. The precipitated pellet was washed thrice with ice-cold 10 mM DTT (Sigma-Aldrich)-acetone (Fisher Scientific) solution and air-dried in a fume hood for 20–30 minutes. The protein pellet was resuspended in 300 μl of solubilization buffer [8 M urea (Sigma-Aldrich), 10 mM DTT (Sigma-Aldrich), 0.1 M triethylammonium bicarbonate (TEAB)(Sigma-Aldrich) at pH 8.5] by vortexing for 20 minutes. The sample was then centrifuged at 15,000 g for 15 min at 20°C twice, and the pellet was discarded.
5
Monomerized Ricin Vaccine Preparation
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Monomerized ricin vaccine was prepared, essentially as previously described [13 (link)]. Briefly, pure ricin was incubated with 50 mM Dithiothreitol (DTT, Sigma-Aldrich, Rehovot, Israel) for 2 h in room temperature, the ricin-DTT solution was incubated for additional 2 h (room temperature, protected from light) with 100 mM Iodoacetamide (IAA, Sigma-Aldrich, Rehovot, Israel), and the product (alkylated-ricin) was extensively dialyzed against PBS.
6
Apoptotic Marker Analysis in C. auris
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For studying apoptotic markers, protoplasts of C. auris MRL6057 were prepared as explained previously [21] (link). Briefly, cells grown for 8 h were then exposed for 4 h to ¼ MIC, ½ MIC and MIC of test compounds. For the purpose of washing and resuspending yeast cells different protoplast buffers (PB) were prepared. After exposure with test compounds, cells were washed and incubated in PB-1 (1 M sorbitol, (Sigma Aldrich Co., USA), 0.05 M tris base (Merck, Germany), 0.01 M MgCl2 (Sigma Aldrich Co., USA), 0.03 M DTT (Merck, Germany), pH 7.4) for 10 min at room temperature. Post-incubation, cells were harvested at 1500 rpm for 5 min and pellet was mixed and incubated in PB-2 (1 M sorbitol, 0.05 M tris base, 0.01 M MgCl2, 0.001 M DTT, pH 7.4) supplemented with lyticase enzyme (1 μg/mL; Sigma Aldrich Co., USA) at room temperature for 1 h. Next, cell suspensions were centrifuged, and pellets were resuspended and incubated in PB-3 (1 M sorbitol, 0.05 M tris base, 0.01 M MgCl2, pH 7.4) at room temperature for 20 min. Post-incubation cell suspension was centrifuged at 1500 rpm for 5 min and pellets with protoplasts were washed and mixed in fresh PBS and stored at 4 °C until further use.
7
Cell Lysis Buffer Preparation
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8
Dietary and Chemical Stress Response in Insects
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Control larvae were allowed to grow either on LSD or HSD diet until pre-wandering. For DTT treatment, three groups of ten LSD-fed larvae were starved 2 h and incubated for 4 h in Schneider’s medium ± 5 mM DTT (Merck #10197777001). Larvae were then washed and frozen in liquid nitrogen. Three groups of ten LSD- or HSD-fed larvae were washed with PBS and frozen simultaneously. RNA extraction and qRT-PCR were performed as described above. Independent experiments were performed using three biological replicates tested in triplicates. For tunicamycin treatment, fat tissues from lpp > xbp1-GFP larvae were incubated 1 h either with 10 ug/ml tunicamycin (1:1000 dilution of 10 mg/ml stock solution, Sigma Aldrich #T7765) or with 1:1000 DMSO in Schneider’s medium at room temperature. Tissues were then washed with PBS and fixed in 37% methanol-free formaldehyde (Thermo Fisher Scientific, #28906). Anti-GFP immunostaining was performed as described above.
9
Extraction of Nuclear Proteins
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Nuclear proteins extracted from HEK cells, SH-SY5Y cells, or brain tissues were used for analyzing THAP1 protein level or for TAP-MS experiments. Briefly, the cytoplasmic fraction was collected using buffer A [10 mM Hepes (Sigma-Aldrich), 1 mM EDTA (Applichem), 0.1 mM EGTA (Sigma-Aldrich), 10 mM KCl (Carl-Roth), 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), phosphatase inhibitor cocktail 2 and 3 (1 μg/ml; Sigma-Aldrich), 1 mM dithiothreitol (DTT; Merck), and complete protease inhibitor (1 μg/ml; Roche)]. After 15 min of incubation, NP-40 (Roche) was added to the final concentration of 0.1% to break the cell membrane. Following centrifugation at 10,000g for 5 min at 4°C, the supernatant was removed (cytoplasmic fraction), and the nuclei were harshly resuspended in buffer C [20 mM Hepes, 0.2 mM EDTA, 0.1 mM EGTA, 25% glycerol (Carl-Roth), 420 mM NaCl (Merck), 1.5 mM MgCl2 (Sigma-Aldrich), 1 mM PMSF, phosphatase inhibitor cocktail 2 and 3 (1 μg/ml), 1 mM DTT, and complete protease inhibitor (1 μg/ml)] and rotated on an intellimixer for 20 min at 4°C. After centrifugation at 10,000g for 5 min at 4°C, the supernatant containing nuclear proteins was collected.
10
In Vitro Fbxw7 Phosphorylation by Cdk5
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GST-tagged WT and mutant variants of human Fbxw7 were expressed in BL21 bacteria with 0.1 mM isopropyl-β-D-thiogalactopyranoside (Sigma–Aldrich) and incubated at 37 °C for 4 h. Bacterial pellets were resuspended in a lysis buffer containing 30 mM Tris-HCl (pH 7.5), 0.1 mM NaCl, 1% Triton X-100 (Sigma–Aldrich), 1 mM DTT (Sigma–Aldrich), and 1 × protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) and then sonicated on ice. Lysates were centrifuged at 12,000 × g for 15 min at 4 °C. The resulting supernatant was incubated overnight at 4 °C with GST beads (GE Healthcare, Chicago, IL). The protein samples were washed three times with 1 × PBS. GST-bound human Fbxw7 protein was incubated with 0.1 μg Cdk5/p35 (Signalchem, Richmond, BC) or Cdk5/p25 (Millipore, Billerica, MA) in a reaction buffer containing 25 mM Tris (pH7.5), 100 mM NaCl, 1 mM DTT, 100 μM unlabeled ATP and 5 μCi [γ-32P] ATP at 30 °C for 30 min. Roscovitine (10 μM) was added 30 min prior to the kinase reaction. The samples were resolved by SDS-PAGE and visualized by autoradiography.
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