Four luminal prostate epithelial cell lines were derived from independent PbCre4; Ptenfl/flTp53fl/fl prostate adenocarcinoma tumors, designated PCAP 1-4 (Prostate Cancer Adenocarcinoma Pten/Tp53 null), and cultured in WIT-P media (Cellaria Biosciences, Cambridge, MA, USA). A normal luminal epithelial prostate cell line (B6WT) was similarly established from a normal C57/BL6 mouse prostate. Human prostate cancer cell lines (PC3, LNCaP, DU145) were originally obtained from ATCC and maintained as described previously. GA, Perminine, Permine, Qurecetin, BBH, Curcumin, Curmunol, Matrine, Oxymatrine, CT, TIIA were purchased from National Institute for Food and Drug Control (Beijing, China). All compounds were prepared as 40mM stocks in DMSO and stored at -20°C in the dark for up to two months. CellTiter-Glo® 96 Aqueous One Solution Cell Proliferation Assay (CTG) kit, Cell Titer-Glo® 3D Cell Viability (CTG-3D) Assay kit and NADPH/NADP Glo assay kit were purchased from Promega (Madison, WI, USA). Antibodies directed against CASP3 (9662), CASP8 (4790), and CASP9 (9508) were obtained from Cell Signaling Co. Ltd (Boston, MA, USA). α-tublin (T8203) antibody was from Sigma (St. Louis, MO, USA). Fluorescent Thioredoxin Activity Assay Kit (20039) and Thioredoxin Reductase Colorimetric Assay Kit (10007892) were purchased from Cayman (Ann Arbor, MI, USA).
Thioredoxin reductase colorimetric assay kit
Manufactured by Cayman Chemical
Sourced in United States
The Thioredoxin Reductase Colorimetric Assay Kit is a laboratory tool used to measure the activity of the enzyme thioredoxin reductase in biological samples. The kit utilizes a colorimetric method to quantify the enzymatic activity.
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Lab products found in correlation
Spark cyto, by Tecan (2 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Nadp nadph glo assay kit, by Promega (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
Glutathione peroxidase assay kit, by Cayman Chemical (1 mentions)
Tissue homogenizer, by Qiagen (1 mentions)
Pierce bca protein kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using thioredoxin reductase colorimetric assay kit
1
Prostate Cancer Cell Line Characterization
2
Redox Enzyme Quantification Assays
3
Thioredoxin Reductase Activity Assay
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TrxR activity was determined using a Thioredoxin Reductase Colorimetric Assay Kit (Cayman, Ann Arbor, MI). Briefly, cells were incubated with GA 500nM for 1and 2H, washed with PBS and homogenized in assay buffer. The supernatant was collected after centrifugation, and equal volumes of total protein were added into a 96-well plate with a master mix containing DTNB and NADPH. Absorbance at 405nm during the initial 15min was recorded in a spectrophotometer. TrxR activity was calculated using the formula provided by the protocol.
4
Thioredoxin Reductase Activity Assay
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The treated and untreated control protein lysates were used to measure TrxR activity, which was determined by using the Thioredoxin Reductase Colorimetric Assay Kit (Cayman Chemical), according to the manufacturer’s protocols. Absorbance was recorded at 405 nm with the Spark®Cyto (Tecan) during the initial 5 min of the reaction. TrxR activity was calculated using the formula provided by the protocol, whereby background measurements were subtracted from all values. An equal amount of protein was loaded for each condition as determined by the Pierce BCA protein kit.
5
Thioredoxin reductase activity assay
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TrxR activity was determined using a thioredoxin reductase colorimetric assay kit (Cayman Chemical). MDA-MB 435 S cells (1.5 × 105/ml) in 60 mm culture dishes were treated with the indicated concentration of AF or Bz for 20 min and then washed with PBS. Cells were harvested with cold 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA and then sonicated. Homogenized cells were centrifuged at 10,000 x g for 15 min at 4 °C and the supernatant was added to the wells of a 96-well plate, along with NADPH and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). TrxR activity was monitored as the ability to use NADPH to reduce 5,5’-dithio-bis(2-dinitrobenzoic acid) to 5-thio-2-nitrobenzoic acid (TNB); this produced a yellow product that was measured at 405 nm. Measurement of TrxR activity by DTNB reduction in the presence and absence of a TrxR inhibitor enabled us to correct non-thioredoxin reductase-independent DTNB reduction.
6
Measuring Thioredoxin Reductase Activity
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After 14 days of AF treatment p.o., SB28 tumors were dissected and disrupted in lysis buffer using a tissue homogenizer (Qiagen, Hilden, Germany). Afterwards, protein lysates were used to measure TrxR activity using the Thioredoxin Reductase Colorimetric Assay kit (Cayman chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Absorbance was recorded at 405 nm with a Spark®Cyto (Tecan, Männedorf, The Switzerland) during the initial 5 min of the reaction. TrxR activity was calculated using the formula provided by the protocol, whereby background measurements were subtracted from all values. An equal amount of protein was loaded for each condition as determined by the Pierce BCA protein kit (Thermo Scientific, Merelbeke, Belgium).
7
Thioredoxin Reductase Activity Assay
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After 24 h of treatment with the tested compounds, thioredoxin reductase activity was measured by Thioredoxin Reductase Colorimetric Assay Kit (Item No. 10007892, Cayman Chemical, Ann Arbor, USA). Cells were homogenized in cold buffer (50 mM potassium phosphate, pH 7.4, containing 1 mM EDTA) and centrifuged (10,000×g, 15 min, 4 °C). The supernatant (20 µl) was used to analyze enzyme activity and to determine protein content. The reaction was initiated by adding 5,5-dithiobis-(2nitrobenzoic acid) (DTNB) solution and NADPH and the change in absorbance caused by the formation of 2-nitro-5thiobenzoic acid (TNB) was measured once every 5 min at 405 nm. Measurement of TrxR activity by DTNB reduction in the absence and in the presence of aurothiomalate (20 µM), allows for correction of non-thioredoxin reductase-independent DTNB reduction. Briefly, the reaction mixture contained 50 mM potassium phosphate (pH = 7.0), 50 mM potassium chloride, 1 mM EDTA, and 0.2 mg/ml BSA. The results were presented as µmol/min/mg of protein.
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