Txnrd1

Manufactured by Abcam

TXNRD1 is a protein-coding gene that provides instructions for making an enzyme called thioredoxin reductase 1. This enzyme plays a crucial role in maintaining the balance of oxidation-reduction (redox) reactions within cells.

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Lab products found in correlation

Gapdh, by Abcam (2 mentions) β actin, by Merck Group (2 mentions) Anti goat horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions) L glutamine, by Nacalai Tesque (1 mentions) Fetal bovine serum (fbs), by Equitech-Bio (1 mentions) Anti rabbit and anti mouse hrp conjugated secondary antibodies, by Cell Signaling Technology (1 mentions) Akr1c1, by Abcam (1 mentions) Akr1c3, by Abcam (1 mentions) Mem nonessential amino acid solution, by Nacalai Tesque (1 mentions) F60 sonic dismembrator, by Thermo Fisher Scientific (1 mentions) Iblot system, by Thermo Fisher Scientific (1 mentions) Criterion tgx precast gel, by Bio-Rad (1 mentions) Lysis buffer, by Cell Signaling Technology (1 mentions) Phospho iκbα, by Abcam (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Total caspase 3, by Cell Signaling Technology (1 mentions) Ripa buffer, by Thermo Fisher Scientific (1 mentions) Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Ai600, by GE Healthcare (1 mentions)

Close spelling variants of this product

Anti-TXNRD1 (4 citations) TXNRD1 activity assay kit (2 citations)

7 protocols using txnrd1

Purification and Evaluation of Isothiocyanates

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ITCs (SFN, 6-MSITC, and 6-MTITC) were purified from Wasabi by reversed-phase high performance liquid chromatography (HPLC) to >99.3% purity26 (link) and dissolved in dimethyl sulfoxide for cell culture experiments. The antibodies against Nrf2 (C-20), Keap1 (E-20), NQO1 (C-19), HSP70 (D69), GAPDH, rabbit IgG, and horseradish peroxidase (HRP)-conjugated anti-goat secondary antibody were purchased from Santa Cruz Biotechnology. AKR1C1, AKR1C3, and TXNRD1 antibodies were obtained from Abcam. HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology.
IMR-32 cell culture. Human neuroblastoma IMR-32 cells (cell no. TKG0207) were obtained from Riken Bioresource Center Cell Bank. IMR-32 cells were grown in Eagle’s Minimum Essential Medium (Nissui Seiyaku) supplemented with 2 mM l-glutamine (Nacalai Tesque), 1% v/v MEM nonessential amino acid solution (Nacalai Tesque), and 10% v/v fetal bovine serum (Equitech-Bio) under a humidified 5% CO₂ atmosphere at 37 °C.

Trio P.Z., Fujisaki S., Tanigawa S., Hisanaga A., Sakao K, & Hou D.X. (2016). DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells. Gene Regulation and Systems Biology, 10, 73-83.

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Western Blot Analysis of Antioxidant Proteins

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Cells were scraped in 1× lysis buffer from Cell Signaling Technology (catalog no. 9803) with protease and phosphatase inhibitors and 0.5% SDS, followed by sonication with F60 Sonic Dismembrator (Fisher Scientific). The amount of protein in each sample was quantified using Pierce BCA Protein Assay Kit. All samples were diluted to the same concentration. For western blot analysis, samples were boiled at 95 °C for 5 min and 20 μg of total protein was loaded on Criterion™ TGX™ precast gels (BioRad Laboratories). Gels were run for 1 h at 150 V followed by transferring proteins onto a nitrocellulose membrane using an iBlot system (Invitrogen). Five percent non-fat dried milk in tris-buffered saline, tween 20 was used to block the membranes for 1 h. After blocking, primary antibodies against GCLC (Abcam), NQO1 (Abcam), HO1 (Abcam), TXNRD1 (Abcam), ACTB (MP Biomedical), Phospho-IκBα (Abcam), and GAPDH (Abcam) were incubated overnight at 4 °C, followed by washing with 1× tris-buffered saline, tween 20 for 30 min. Secondary antibodies were then incubated at room temperature for 45 min. After washing the membranes for 30 min, Super Signal West Femto Substrate was added to detect horseradish peroxidase on the membranes. Chemiluminescence was measured and quantified using the Syngene gel imaging system.

Patel H., McIntire J., Ryan S., Dunah A, & Loring R. (2017). Anti-inflammatory effects of astroglial α7 nicotinic acetylcholine receptors are mediated by inhibition of the NF-κB pathway and activation of the Nrf2 pathway. Journal of Neuroinflammation, 14, 192.

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Investigating AF-Induced Apoptosis Signaling

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Cells were treated with AF at various concentrations for 12 h and protein extracts perepared. Samples were resolved using 10% SDS-PAGE. Proteins were detected with antibodies to TXNRD1 1:1000 (Abcam); TXNRD2 1:1000; β-actin 1:6,000 (all from Sigma-Aldrich); BIM 1:1000; BAD 1:1000; Cleaved caspase-3 1:1000; Total caspase-3 1:1000 (all from Cell Signaling). Horseradish peroxidase conjugated secondary goat anti-rabbit (Santa Cruz) and rabbit anti-mouse (BD Pharmingen) were used for detection.

Topkas E., Cai N., Cumming A., Hazar-Rethinam M., Gannon O.M., Burgess M., Saunders N.A, & Endo-Munoz L. (2015). Auranofin is a potent suppressor of osteosarcoma metastasis. Oncotarget, 7(1), 831-844.

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Western Blot Analysis of Oxidative Stress Markers

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Cells were exposed or transfected as previously described and washed with PBS and lysed in ice-cold RIPA buffer (ThermoFisher) with protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined with the BCA Protein Assay Kit (Pierce) and samples were boiled with 4X Laemmli buffer for 5 min. Equal amounts of protein were resolved in 4%–15% gradient SDS-PAGE gels (BioRad) and transferred to polyvinylidene difluoride membranes (Merck Millipore). Membranes were cut according to target molecular weight and probed with anti-NRF2 (Abcam, #137550), TXNRD1 (Abcam, #124954), or GAPDH (Abcam, #245355) overnight on a shaker at 4°C. Antibody dilutions are presented in Supplementary Table S2. Membranes were washed 3X in 5% non-fat milk for 15 min each before incubating for 1 h on a shaker at room temperature in secondary antibody (Abcam, #7090). Finally, membranes were washed 3X in TBST for 15 min each, incubated in enhanced chemiluminescence reagents (ThermoFisher), and imaged with the AI600 (GE). Band density analysis was performed using Photoshop (Adobe).

Simmers M.D., Jima D.D., Tsuji Y, & Cowley M. (2023). LncRNA Tuna is activated in cadmium-induced placental insufficiency and drives the NRF2-mediated oxidative stress response. Frontiers in Cell and Developmental Biology, 11, 1151108.

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Western Blotting Characterization of Proteins

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Western blotting was performed as previously described. Briefly, 2×10⁶ cells were harvested with medium flow, pelleted by centrifugation for 5 min at 500 g, and washed 3 times with PBS. The cells were then lysed with 100 µl lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1.5% NP-40, 0.1% SDS, and 50 µg/ml PMSF, with freshly added proteinase inhibitor cocktail) on ice for 30 min. Lysates were centrifuged at 13,000 rpm for 15 min. and the protein concentration was measured using Bradford methods. Fifty micrograms proteins was separated on 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA in TBST for one hour at room temperature followed by incubation with primary antibody solutions according to the manufacturer's instructions (β-actin, 1∶5000, Sigma; NAIF1, 1∶1000, Santa Cruz; 14-3-3 ε, 1∶500, Santa Cruz; TCP1-γ, 1∶1000, Abcam; TXNRD1, 1∶1000, Abcam; CyclinD1, 1∶1000, CST; cdc2, 1∶500, CST; and p21, 1∶500, CST). The membranes were washed 3 times with TBST, followed by incubation with appropriate secondary antibodies for two hours at room temperature. The signals were detected using the ECL Chemiluminescence system. β-actin was employed as the control.

Yang M., Zhong J., Zhao M., Wang J., Gu Y., Yuan X., Sang J, & Huang C. (2014). Overexpression of Nuclear Apoptosis-Inducing Factor 1 Altered the Proteomic Profile of Human Gastric Cancer Cell MKN45 and Induced Cell Cycle Arrest at G1/S Phase. PLoS ONE, 9(6), e100216.

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Evaluation of Antioxidant Pathways in Organoids

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Organoid cultures were harvested at the indicated time points using dispase, washed with PBS and lysed in Laemmli lysis buffer (2.5% SDS, 20% glycerol, 120 mM TRIS pH6.8). Equal amounts of protein (10-20 mg) were run on SDS-PAA gels transferred onto nitrocellulose membranes (Trans-Blot Turbo, Bio-Rad, Hercules, CA, USA), and incubated with antibodies as described. Primary antibodies to TXNRD1, GCLC, GCLM, and Glutathione Syntehase (GS) were all purchased from Abcam. GPX2 antibody was a (Kind gift from Anna Kipp (Friedrich Schiller University Jena, FSU. Institute of Nutrition, Germany). Phospho-Mek1/2, phospho-Erk1/2 (Thr202/ Tyr204) and phospho-AKT (Ser473) (163H12) were all from Cell Signaling. Phospho-NRF2 phospho (pS40) mAb was purchased from (ABGent), p53 antibody (Santa Cruz antibodies), and b-actin (Novus). Table S5 gives an overview of antibodies and dilutions.

Laoukili J., van Schelven S., Küçükköse E., Verheem A., Goey K., Koopman M., Borel Rinkes I, & Kranenburg O. (2022). BRAF^V600E in colorectal cancer reduces sensitivity to oxidative stress and promotes site-specific metastasis by stimulating glutathione synthesis. Cell reports, 41(9).

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Analyzing Apoptosis and Oxidative Stress

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Protein expression of apoptotic and oxidative stress markers were measured in the cytosolic fraction collected for GR quantification. 20 µg of total protein was loaded into 15% polyacrylamide gels and transferred to low fluorescence PVDF membranes and incubated overnight with the following apoptosis-related primary antibodies; BCL2 (1:1000, Abcam, Cat#ab32124), BAX (1:000, Cell Signaling, Cat#2772) and cleaved caspase 3 (1:1000, Cell Signaling, Cat#9661) as well as primary antibodies for the antioxidant proteins thioredoxin reductase (Txnrd1, 1:1000, Abcam, Cat#ab124954) and glutathione peroxidase (GPX, 1:1000, Abcam, Cat#ab108427). After incubation with the primary antibody of interest, all membranes were incubated with a ACTB loading control (1:20,000, Cat# A2228, Sigma Aldrich) for 1 h. After careful washing, membranes were incubated with antimouse and anti-rabbit fluorescent secondary antibodies for 1 h. Membranes were then scanned using the Licor Odyssey and densitometry determined using the 234:2 supplied software. Total protein levels of the proteins of interest were normalized to the ACTB loading control.

Cuffe J.S.M., Saif Z., Perkins A.V., Moritz K.M, & Clifton V.L. (2017). Dexamethasone and sex regulate placental glucocorticoid receptor isoforms in mice. The Journal of endocrinology, 234(2).

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