HEK293 cells (ATCC CRL-1573) were transiently transfected with mWNK4-HA35 (link) or hKLHL3-FLAG34 (link). Cells were grown to 70–80 % confluence and transfected with Lipofectamine 2000 (Life Technologies). Forty-eight hours later, cells were lysed with a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM ethyleneglycol-bis-(b-aminoethylether)-N,N,N0,N0-tetra-acetic acid, 1 mM ethylenediamine tetra-acetic acid, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% (w/v) IGEPAL® CA-630 (Sigma), 270 mM sucrose, 0.1% (v/v) 2-mercaptoethanol, and protease inhibitors (complete tablets, 10 mM 1,10-phenanthroline, and 1 mg/ml pepstatin; Roche).
2 mercaptoethanol
Manufactured by Roche
Sourced in Switzerland
2-mercaptoethanol is a colorless, viscous liquid chemical compound. It is commonly used in biochemical and molecular biology applications as a reducing agent to break disulfide bonds and maintain proteins in a reduced state.
Automatically generated - may contain errors
Lab products found in correlation
Igepal ca 630, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Pepstatin, by Roche (1 mentions)
Sodium dodecyl sulphate (sds), by Serva Electrophoresis (1 mentions)
Glucanex, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Serva Electrophoresis (1 mentions)
Ribonuclease a, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Proteinase k, by Roche (1 mentions)
2 protocols using 2 mercaptoethanol
1
Transient Transfection of HEK293 Cells
2
Enzymatic DNA Extraction from Fungal Spores
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About 10–20 mg of spores were enzymatically treated for 2 hours at 37°C in 150 μl solution of 10 mg·ml-1 Lysing Enzymes from Trichoderma harzianum (syn. Glucanex®, Sigma-Aldrich, USA), 1% Triton X-100 (Sigma-Aldrich, USA), 4% 2-mercaptoethanol (Sigma-Aldrich, USA), and 50 mmol·l-1 EDTA (Sigma-Aldrich, USA), pH 5.6. Samples were stirred several times during the treatment. Subsequently, 100 μl of lysis solution consisting of 500 mmol·l-1 NaCl, 100 mmol l-1 Tris-HCl, 50 mmol·l-1 EDTA (pH 8.0), 0.02% Sodium Dodecyl Sulphate, (Serva, Germany), 0.5% w/v L-ascorbic acid (Sigma-Aldrich, USA), 0.03% w/v proteinase K (Roche Diagnostics, Switzerland) and 4% 2-mercaptoethanol was added to the suspension. The lysis was carried out by incubation 45 min at 65°C. Isolated DNA was then purified by phenol-chloroform extraction. Finally, the DNA was precipitated with isopropanol and dissolved in sterile deionized water. RNA was eliminated from the samples by incubation 20 min at 37°C with 10 mg·l-1 ribonuclease A (Sigma-Aldrich, USA).
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