Cells were grown in 6-well culture plates and treated with BBSKE, oxaliplatin, or the combination for the indicated times. Next, the cells were lysed with lysis buffer and protein concentrations were determined using the Bradford assay. TrxR1 activity in cell lysates or tumor tissues was determined using an endpoint insulin reduction assay [34 (link)]. Briefly, cell extracts containing 100 µg total protein were incubated in a final reaction volume of 50 µL containing 0.3 mM insulin, 100 mM Tris-HCl (pH 7.6), 3 mM EDTA, 660 µM NADPH, and 15 µM E. coli Trx (Sigma, St. Louis, MO) for 30 min. The reactions were terminated by adding 200 µL 1 mM DTNB in 6 M guanidine hydrochloride (pH 8.0). A blank control sample (containing everything except Trx) was treated in the same manner. The absorbance was measured using a SpectraMax iD3 microplate reader (Molecular Devices, USA) at 412 nm, and the activity was expressed as percentage of the blank control.
E coli trx
Manufactured by Merck Group
Sourced in Macao
E. coli Trx is a laboratory product that functions as a thioredoxin protein derived from the Escherichia coli bacterium. Thioredoxins are small, ubiquitous proteins that play a crucial role in redox regulation and protein folding within cells.
Automatically generated - may contain errors
4 protocols using e coli trx
1
Thioredoxin Reductase 1 Activity Assay
2
Determination of TrxR Activity in Cell Lysates
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After the treatment of cells with IBC with different concentrations for two hours, the harvesting and extraction of cells were achieved with RIPA buffer. The Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) was applied to determine the content of total protein. Based on an insulin decrease assay at the end points, the measurement of TrxR activity in cell lysates was achieved. Soon, incubation of cell extract was achieved and the eventual reaction volume was 50 μL, containing 100 mM Tris-HCl (pH 7.6), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA, and 15 μM E. coli Trx (Sigma-Aldrich, St. Louis, MO), for a duration of 30 min at 37°C. A total of 40 μg of protein were contained in the extract. Through the addition of 200 μL of 1 mM DTNB in 6 M guanidine hydrochloride (pH 8.0), the termination of the reaction was achieved. A blank sample, containing everything except Trx, was treated in the same manner.
The measurement of absorbance at 412 nm was carried out, followed by the subtraction of the blank value from the absorbance value of the sample accordingly. The activity was denoted as the percentage of the subject in the control group.
The measurement of absorbance at 412 nm was carried out, followed by the subtraction of the blank value from the absorbance value of the sample accordingly. The activity was denoted as the percentage of the subject in the control group.
3
Measuring Thioredoxin Reductase Activity
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After cells were treated with various concentrations of EF24 for 2 h, the cells were harvested and protein extracted with RIPA buffer. The total protein content was determined by using the Bradford protein assay kit (Bio-Rad, Hercules, CA). TrxR activity in cell lysates or tumor tissues was measured by the end point insulin reduction assay. Briefly, the cell extract containing 100 μg of total proteins was incubated in a final reaction volume of 50 μL containing 100 mM Tris-HCl (pH 7.6), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA, and 15 μM E. coli Trx (Sigma, St. Louis, MO) for 30 min at 37°C. The reaction was terminated by adding 200 μL of 1 mM DTNB in 6 M guanidine hydrochloride (pH 8.0). A blank sample, containing everything except Trx, was treated in the same manner. The absorbance at 412 nm was measured, and the blank value was subtracted from the corresponding absorbance value of the sample. The activity was expressed as the percentage of the control.
4
Reactivation of Glutathionylated AtAMY3
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Freshly reduced AtAMY3, obtained as previously described, was incubated with 5 mM GSSG for 90 min at 25°C. After GSSG incubation, sample was desalted through NAP-5 column (GE Healthcare) in 100 mM Tricine-NaOH, pH 7.9 and brought to the desired concentration (20 μM) through Amicon-Ultra device (Millipore; cut-off 10 kDa). Sample concentration was determined by absorbance at 280 nm (Nanodrop; Thermo Fisher Scientific).
Reactivation treatments of glutathionylated AtAMY3 were performed by incubating samples for 30 min at 25°C in the presence of 50 μM, 0.2 mM, 1 mM, 2 mM, 5 mM, and 7 mM GSH. Samples were then diluted 5-fold and the activity assayed as described above.
Reactivation assays of GSSG-inhibited AtAMY3 performed in the presence of poplar GRX S12 and A. thaliana GRX C5, and commercially available E. coli TRX (Sigma-Aldrich; protein id AAA24693), were conducted through 5, 15, and 30 min of incubation of 20 μM glutathionylated AtAMY3 in the presence of 2 mM GSH with or without 5 μM GRXs, or in the presence of 0.2 mM DTT with or without 10 μM TRX. Upon incubation, enzyme activity was measured on 5-fold diluted samples as described above.
All data are expressed as percentages of activity relative to the fully reduced sample obtained by incubating glutathionylated AtAMY3 in presence of 80 mM DTT for 30 min at 25°C.
Reactivation treatments of glutathionylated AtAMY3 were performed by incubating samples for 30 min at 25°C in the presence of 50 μM, 0.2 mM, 1 mM, 2 mM, 5 mM, and 7 mM GSH. Samples were then diluted 5-fold and the activity assayed as described above.
Reactivation assays of GSSG-inhibited AtAMY3 performed in the presence of poplar GRX S12 and A. thaliana GRX C5, and commercially available E. coli TRX (Sigma-Aldrich; protein id AAA24693), were conducted through 5, 15, and 30 min of incubation of 20 μM glutathionylated AtAMY3 in the presence of 2 mM GSH with or without 5 μM GRXs, or in the presence of 0.2 mM DTT with or without 10 μM TRX. Upon incubation, enzyme activity was measured on 5-fold diluted samples as described above.
All data are expressed as percentages of activity relative to the fully reduced sample obtained by incubating glutathionylated AtAMY3 in presence of 80 mM DTT for 30 min at 25°C.
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