Ab126704

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab126704 is a primary antibody, specifically a rabbit monoclonal [EPR16571] to Heat Shock Protein 90 (HSP90), a molecular chaperone protein involved in protein folding and stabilization. The antibody is intended for use in various immunoassay applications to detect and quantify HSP90.

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Lab products found in correlation

11 protocols using ab126704

Redox-Sensitive Protein Regulation Protocol

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The chemical (2S, 3S)-1,4-Bis-sulfanylbutane-2,3-diol (DTT, CAS No. 3483-12-3) was obtained from Sigma; it is a strong reductant with the chemical formula C₄H₁₀O₂S₂ and MW of 154.25 g/mol. Its reducibility is largely due to the conformational stability after oxidation (containing disulfide bond). In this experiment, 3.09 g DTT powder was completely dissolved in 20 mL 0.01 M sodium acetate to obtain 1 M DTT stock solution, which was packed and stored at −20 °C before use.
Specific antibody against Nrf1 was made in our own laboratory [25 (link)]. All nine distinct antibodies against Nrf2 (ab62352), GCLC (ab207777), GCLM (ab126704), HO-1 (ab52947), GPX1 (ab108427), XBP1 (AB109221), ATF4 (ab184909), ATF6 (ab227830) and P4HB (ab137180) were obtained from Abcam (Cambridge, UK). The first three antibodies against TALDO (D623398), GSR (D220726) and NQO1 (D26104) were purchased from Sangon Biotech (Shanghai, China). The second three antibodies against BIP (bs-1219R), Chop (bs-20669R) and pIRE1 (bs-16698R) were from Bioss (Beijing, China). The third three antibodies against PSMB5 (A1975), PSMB6 (A4054), PSMB7 (A14771) were from ABclonal (Wuhan, China). Lastly, three antibodies to p-eIF2α (#5199) were from Cell Signaling Technology (Boston, MA, USA), p-PERK (sc-32577) from Santa CruZ (Santacruz, CA, USA), and β-actin (TA-09) from ZSGB-BIO (Beijing, China), respectively.

Wufuer R., Fan Z., Yuan J., Zheng Z., Hu S., Sun G, & Zhang Y. (2022). Distinct Roles of Nrf1 and Nrf2 in Monitoring the Reductive Stress Response to Dithiothreitol (DTT). Antioxidants, 11(8), 1535.

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Oxidative Stress Regulation in TDSCs

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TDSCs were treated with H₂O₂ or pretreated with PCs for 24 h as for Real-Time PCR. Afterward, TDSCs were washed with PBS, lysed in lysis buffer, and kept on ice for 5 min by Whole Protein Extract Kit (Jiancheng Bioengineering, Nanjing). Cell lysates were centrifuged at 20,000 rpm at 4°C for 5 min, and the supernatants were stored at −80°C until use. Protein concentrations were measured by using a protein assay kit (Jiancheng Bioengineering, Nanjing). Twenty micrograms of total protein were diluted in loading buffer, separated by SDS/PAGE, and electroblotted onto PVDF membranes. The membranes were then blocked with TBS-Tween 20 solution containing 5% nonfat dry milk and incubated overnight at 4°C with specific antibodies against Nrf-2 (1 : 200) (ab31163, Abcam, USA), GCLM (1 : 1000) (ab126704, Abcam, USA), HO-1 (1 : 200) (ab68477, Abcam, USA), and β-actin (ab8226, Abcam, USA). Proteins were visualized using goat anti-rabbit conjugated to HRP and a Chemiluminescence Western Blotting Detection system (34079, Thermo Pierce, USA). Protein band intensities were quantified using the Quantity One software.

Sun W., Meng J., Wang Z., Yuan T., Qian H., Chen W., Tong J., Xie Y., Zhang Y., Zhao J, & Bao N. (2017). Proanthocyanidins Attenuation of H2O2-Induced Oxidative Damage in Tendon-Derived Stem Cells via Upregulating Nrf-2 Signaling Pathway. BioMed Research International, 2017, 7529104.

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Western Blot Analysis of DNA Damage Markers

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Cells were directly lysed using 2 × protein lysing buffer (2% SDS, 5% β-mercaptoethanol, 0.5% sucrose and 0.2% bromophenol blue) at 4°C for 20 min, and then the lysates were heated in a metal bath at 98°C for 5 min. Cell lysates were separated on 4–12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked using 5% non-fat milk and immunoblotted using indicated antibodies. The protein signals were detected using a New-SUPER ECL Substrate Kit (Keygen Biotech, Nanjing, China). The primary antibodies used in this study targeted phospho-ATR (S428) (1:1000, abcam, ab178407), phospho-ATM (S1981) (1:1000, Cell Signaling Technology, #5883), phospho-p53 (S15) (1:1000, abcam, ab223868), phospho-histone H2AX (S139) (1:1000, Cell Signaling Technology, #9508), GCLM (1:1000, abcam, ab126704), HO-1 (1:1000, abcam, ab68477), phospho-p38 (T180/Y182) (1:500, Santa Cruz Biotechnology, sc-17852-R), phospho-Hsp27 (S82) (1:1000, abcam, ab155987), phospho-ATF2 (T71) (1:1000, abcam, ab32019) and Actin (1:3000, Bioss, #bs-0061R). The secondary antibodies used in this study were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:10,000, TransGen Biotech, Beijing, China, #HS201-01) and HRP-conjugated goat anti-rabbit IgG (1:10,000, TransGen Biotech, Beijing, China, #HS101-01).

He H., Zou Z., Wang B., Xu G., Chen C., Qin X., Yu C, & Zhang J. (2020). Copper Oxide Nanoparticles Induce Oxidative DNA Damage and Cell Death via Copper Ion-Mediated P38 MAPK Activation in Vascular Endothelial Cells. International Journal of Nanomedicine, 15, 3291-3302.

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Autophagy and copper regulation in cells

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The following antibodies against GCLM (1:2000; ab126704), p62 (1:2000; ab109012), ATG5 (1:3000; ab109409), ATG7 (1:10,000; ab52472), LC3B (1:3000; ab192890), p-mTOR (1:2000; ab109268) were purchased from Abcam (Cambridge, UK). The following antibodies against BIP (1:1000; 3177), mTOR (1:1000; 2983), p-AMPK α (1:2000; 2573S), AMPKα (1:2000; 5831), Beclin1 (1:1000; 3495) were purchased from Cell Signaling Technology (Danvers, MA). The CD68 (1:200; 28058-1-AP) and HO-1 (1:1000; 10701-1AP) was purchased from Proteintech (Chicago, USA). The ULK1 (1:1000; A5149) was purchased from Bimake (Houston, USA). The β-actin Ab was obtained from Bioss (1:5000; bs-0061R) (Beijing, China). The QuantiChrom^TM Copper Assay Kit (DICU-250) was purchased from Bioassay (California, USA).

Xiao J., Tu B., Zhou X., Jiang X., Xu G., Zhang J., Qin X., Sumayyah G., Fan J., Wang B., Chen C, & Zou Z. (2021). Autophagy deficiency exacerbates acute lung injury induced by copper oxide nanoparticles. Journal of Nanobiotechnology, 19, 162.

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Comprehensive Glutathione Pathway Analysis

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All chemicals, unless specified, were purchased from Sigma. Antibodies against glutathione [D8] (ab19534), GCLC (ab53179), GCLM [EPR6667] (ab126704), glutathione synthetase [EPR6562] (ab124811), glutathione reductase (ab16801), xCT or Slc7a11 (ab37185), PPARα (ab24509), PPARγ (ab19481), PGC1α (ab54481), Vitamin D receptor (ab3508), and β-actin HRP (ab49900) were purchased from Abcam. The anti-CTH (WH0001491M) and anti-GLUT4 (G4048) were purchased from Sigma Aldrich. Goat anti- mouse HRP (170–6516) was purchased from Biorad and the goat anti-rabbit HRP (12–348) from Millipore. Pierce^™ protein A/G agarose was purchased from Thermo Scientific.

Parsanathan R, & Jain S.K. (2018). Hydrogen sulfide increases glutathione biosynthesis, and glucose uptake and utilization in C2C12 mouse myotubes. Free radical research, 52(2), 288-303.

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Quantifying Antioxidant Enzyme Levels

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Western blot analysis was performed using lysates from astrocytes. The blots were probed with antibodies against NQO1 (Sigma N5288), GCLC (Abcam ab53179), GCLM (Abcam ab126704) and β-actin (Abcam ab8227) using the dilutions recommended in the product datasheet. Band densities were analyzed with ImageJ software.

Bakshi R., Zhang H., Logan R., Joshi I., Xu Y., Chen X, & Schwarzschild M.A. (2015). Neuroprotective effects of urate are mediated by augmenting astrocytic glutathione synthesis and release. Neurobiology of disease, 82, 574-579.

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Nrf2 Pathway Regulation Assay

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All chemicals were of the highest quality commercially available. The 26S proteasome inhibitor (MG132, bortezomib) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies such as V5 (Invitrogen, Shanghai, China), KEAP1 (D154142, Sangon Biotech, Shanghai, China), as well as Nrf2 (ab62352) and its target HO1 (ab52947), GCLM (ab126704) and NQO1 (ab80588) (all the latter four antibodies from Abcam, Shanghai, China) were herein employed. In addition to another first antibody against β-actin, various secondary antibodies were obtained from ZSGB-BIO (Beijing, China).

Qiu L., Wang M., Zhu Y., Xiang Y, & Zhang Y. (2018). A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2. International Journal of Molecular Sciences, 19(8), 2150.

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Antioxidant Protein Expression in PC12 Cells

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PC12 cells were incubated in six‐well plates at a density of 2 × 10⁵ and incubated for 24 h at 37 °C. After suitable treatments, the cells were collected and lysed with ice‐cold RIPA lysis buffer. Equal amounts of protein samples (80 μg) were separated on a 10% SDS/PAGE gel and transferred electrophoretically onto nitrocellulose membrane. Subsequently, the membranes were blocked with 5% skim milk and incubated overnight at 4 °C with primary antibody: anti‐HO‐1 (ab68477; Abcam Biotechnology, Cambridge, MA, USA), anti‐GCLC (ab190685; Abcam Biotechnology), anti‐GCLM(ab126704; Abcam Biotechnology), anti‐NQO‐1 (ab124954; Abcam Biotechnology), anti‐TrxR‐1 (ab124954; Abcam Biotechnology) and anti‐β‐actin (AP0060; Bioworld Technology, Bloomington, MN, USA). After being washed three times with 1× Tis‐buffered saline‐Tween 20, the membrane was incubated with anti‐rabbit IgG for 1 h at room temperature. Proteins were visualized by exposure in a ChemiDoc XRS + system (Bio‐Rad, Hercules, CA, USA). Band density was quantified by imagej (National Institute of Health, Bethesda, MD, USA).

Zhang Y., Wang Z., Yang J., He Y., Wan H, & Li C. (2021). Analogs of imine resveratrol alleviate oxidative stress‐induced neurotoxicity in PC12 cells via activation of Nrf2. FEBS Open Bio, 11(8), 2127-2138.

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Immunoblot Assay for Redox Proteins

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The immunoblot assays were performed as described.^[⁶⁴, ⁶⁶, ⁶⁷^] Briefly, whole cell lysates or tumor tissues were prepared in lysis buffer supplemented with protease and phosphatase inhibitors (Roche). The lysates were centrifuged at 12 000 × g for 10 min at 4 °C followed by quantification with the BCA Protein Assay Kit (Thermo Fisher Scientific, 23 225). Equal amounts of protein were subject to SDS‐PAGE followed by transfer and immunoblot. Primary antibodies were anti‐GCLM (ab126704, Abcam), anti‐GSR (A12070, ABclonal), anti‐GPX4 (ab125066, Abcam), anti‐G6PD (HPA000834.Sigma), anti‐Tubulin (KM9003, Sungene Biotech), and secondary antibodies were HRP‐conjugated goat‐anti‐mouse or rabbit IgG (Thermo Fisher Scientific, PA1‐86717, and SA1‐9510, respectively).

Wang P., Yang W., Guo H., Dong H., Guo Y., Gan H., Wang Z., Cheng Y., Deng Y., Xie S., Yang X., Lin D, & Zhong B. (2021). IL‐36γ and IL‐36Ra Reciprocally Regulate NSCLC Progression by Modulating GSH Homeostasis and Oxidative Stress‐Induced Cell Death. Advanced Science, 8(19), 2101501.

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Protein Extraction and Analysis

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All chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to GCLc (ab240379), GCLm (ab126704), Band 3 (ab108414), GAPDH (ab9485), and phosphotyrosine (ab179530) were obtained from Abcam (Cambridge, MA). Electrophoresis equipment and related supplies were obtained from Bio-Rad (USA). The spectrophotometer was purchased from Thermo Fisher Scientific (USA).

Wang Y., Zhao N., Xiong Y., Zhang J., Zhao D., Yin Y., Song L., Yin Y., Wang J., Luan X, & Xiong Y. (2020). Downregulated Recycling Process but Not De Novo Synthesis of Glutathione Limits Antioxidant Capacity of Erythrocytes in Hypoxia. Oxidative Medicine and Cellular Longevity, 2020, 7834252.

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