CoNPs (< 50 nm, #7440-48-4), alpha-lipoic acid (#1077-28-7), and dichlorofluorescin diacetate (DCFH-DA) (#4091-99-0) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and trypsin–EDTA were procured from Gibco (Life Technologies, Paisley, UK). CCK-8 kits were obtained from Dojindo (Kumamoto, Japan). Annexin V-FITC (#AP101-100-AVF), Calcein AM/PI (#C2013FT& ST511) was purchased from Multi Sciences (Hanzhou, China). DMSO, GSH, and GSSG Assay Kit (#S0053) were procured from Beyotime (Shanghai, China). Iron Assay Kit (#ab83366), Anti-GPx4 antibody (#ab125066), Goat anti-rabbit IgG H&L (HRP) (ab205718), Goat anti-mouse IgG H&L (HRP) (ab205719), and β-actin antibody were obtained from Abcam Technology (Cambridge, UK).
Anti gpx4 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-GPX4 antibody is a laboratory tool used to detect and study the glutathione peroxidase 4 (GPX4) protein. GPX4 is an enzyme involved in the regulation of oxidative stress in cells. This antibody can be used in various research applications, such as western blotting, immunohistochemistry, and immunofluorescence, to investigate the expression and localization of GPX4 in different biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Iron assay kit, by Abcam (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Cck 8 kit, by Dojindo Laboratories (1 mentions)
Dichlorofluorescin diacetate dcfh da, by Merck Group (1 mentions)
Conps, by Merck Group (1 mentions)
Goat anti mouse igg h l hrp ab205719, by Abcam (1 mentions)
Gssg assay kit, by Beyotime (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
β actin antibody, by Abcam (1 mentions)
Dimethyl sulfoxide (dmso), by Beyotime (1 mentions)
Glutathione (gsh), by Beyotime (1 mentions)
Anti p53 antibody, by Abcam (1 mentions)
Polyclonal anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Odyssey system, by LI COR (1 mentions)
Anti mouse secondary antibody, by Wuhan Servicebio Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Bca assay kit, by Solarbio (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
8 protocols using anti gpx4 antibody
1
CoNPs Cytotoxicity and Antioxidant Assays
2
Protein Expression Profiling in HK-2 Cells
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Western blotting was performed to determine the expression of inositol-requiring protein 1 (IRE1) p-IRE1/IRE1, eIF2, p-eIF2, PERK, p-PERK, transcription factor 4 (ATF-4), transcription factor 6 (ATF-6), C/EBP-homologous protein (CHOP), p53, cystine glutamate transporter (SLC7A11), and glutathione peroxidase (GPX4) in HK-2 cells. Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Grand Island, NY, USA). Proteins of HK-2 cells were separated by SDS PAGE and then transferred electrophoretically onto polyvinylidenedifluoride (PVDF) membranes. The membranes were probed with anti-p-IRE1/IRE1 antibody, anti-ATF-4/6 antibody anti-CHOP antibody, anti-P53 antibody, anti-SLC7A11 antibody, anti-GPX4 antibody (Abcam, Cambridge, MA, USA), and polyclonal anti-actin antibodies (Santa Cruz Biotechnology, Dallas, USA). Protein bands were detected using an Odyssey System from LI-COR Biosciences, USA.
3
Western Blot Analysis of Ferroptosis Markers
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Proteins from cell samples and colon tissues were extracted using RIPA lysis mixed with PMSF (Solarbio, Beijing, China) and quantified using a BCA assay kit (Solarbio, Beijing, China). Then, the total protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking with 5% skim milk, PVDF membranes were incubated with anti-GPX4 antibody (dilution at 1 : 1000, Abcam, Cambridge, UK), anti-ACSL4 antibody (dilution at 1 : 1000, Abclonal, Wuhan, China), and anti-GAPDH antibody (dilution at 1 : 1000, Sevicebio, Wuhan, China) at 4°C overnight. On the other day, the membranes were incubated with anti-mouse secondary antibody (dilution at 1 : 1000, Servicebio, Wuhan, China) or anti-rabbit secondary antibody (dilution at 1 : 1000, CST, Boston, USA) for 50 min at room temperature. Finally, signals were detected with electrochemiluminescence solution (ECL, Millipore, Massachusetts, USA) using a ChemiScope exposure machine (Clinx Science Instruments Co., Ltd., Shanghai, China) and images were analyzed by ImageJ software.
4
Ferroptosis Regulation with O-CMC Nanoparticles
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SIM and lecithin were provided by Macklin (Shanghai, China). O-carboxymethyl chitosan (O-CMC) was supplied by Santa Cruz (TX, USA). Perfluorohexane (PFH) was achieved from Aladdin (Shanghai, China). Tween 20 was supplied by Solarbio (Beijing, China). Mevalonic acid was achieved from Sigma-Aldrich (St Louis, MO, USA). ROS inhibitor (NAC) was obtained from Beyotime (Shanghai, China). Matrigel was achieved from Corning (New York, USA). An anti-GPX4 antibody was supplied by Abcam (Cambridge, UK). An anti-GAPDH antibody was supplied by BOSTER (Wuhan, China). Anti-Ki67 antibody was supplied by Servicebio (Wuhan, China).
5
Ferroptosis Modulation in Cell Lines
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Erastin, rapamycin, z-VAD-fmk, 3-MA, and DFO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fer-1, Lip-1, and Baf-A1 were purchased from Selleck Chemicals (Houston, TX, USA). BSO was purchased from Cayman Chemical (Ann Arbor, MI, USA). Anti-LC3B (D11), anti-GAPDH antibodies, and senescence β-galactosidase staining kit were purchased from Cell Signaling Technology (Danvers, MA, USA). Micro BCA Protein Assay Kit, H2DCFDA (H2-DCF, DCF), C11-BODIPY (581/591), MitoSOX™ Red Mitochondrial Superoxide Indicator, MitoTracker® Red CMXRos, and Dulbecco’s modified Eagle’s medium (High glucose, no glutamine, no methionine, no Cys2) were purchased from Thermo Scientific (Waltham, MA, USA). Dulbecco’s modified Eagle’s medium/F12 and fetal bovine serum were purchased from Gibco (Logan, UT, USA). Anti-p16-INK4A antibodies was purchased from Proteintech (Rosemont, IL, USA). Alexa Fluor® 488 annexin V/dead cell apoptosis kit was purchased from Invitrogen (Carlsbad, CA, USA). Anti-GPX4 antibody and propidium Iodide flow cytometry kit were purchased from Abcam (Cambridge, MA, USA). GSH assay kit was purchased from Beyotime Biotechnology (Nantong, Jiangsu, China).
6
Western Blot Analysis of Protein Markers
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Western blot analysis was performed as previously described [6 (link), 29 (link)]. The antibodies were diluted in 3 % blocking agent: anti-phospho-p38 MAPK Antibody (1:1000, Cell Signaling, Danvers, MA), Anti-PI3K p85 (phosphor Y607) antibody (1:1000, Abcam, Cambridge, UK), anti-phospho-PKA substrate antibody (1:10,000; Cell Signaling Technology, MA, USA), anti-phosphotyrosine antibody (PY20, 1:2,500, Abcam), anti- GAPDH antibody (1:1000, Abcam), anti-UQCRFS1 antibody (1:15,000, LSBio, Inc.), anti- PRDX5 antibody (1:2000, Abcam), anti-GPX4 antibody (1:15,000, Abcam), anti-ACTB antibody (1:500, Abcam), and anti-GSTM5 antibody (1:5,000, Abcam). Anti-α-tubulin mouse antibody (1:1000, Abcam) was used as the loading control for all western blots. The proteins on the membranes were detected with by an enhanced chemiluminescence (ECL) technique using ECL detection reagents.
7
DOR Agonist Effects on Ferroptosis
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For cell cultures, we obtained RPMI 1640 medium and fetal bovine serum (FBS) from Sigma-Aldrich (St. Louis, MO, USA). [d-Ala2, d-Leu5]-Enkephalin (DADLE), a specific DOR agonist; naltrindole, a specific DOR antagonist and ferrostatin-1, a ferroptosis inhibitor, were purchased from MedChemExpress Co. (Shanghai, China). The antibodies for western blot analysis were purchased from Abcam Co. (St. Louis, MO, USA): antibeta actin antibody, anti-Nrf2 antibody, anti-GPX4 antibody, and anti-xCT antibody.
8
Immunofluorescent Analysis of GPX4 Expression
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The HUVECs were fixed with 4% paraformaldehyde for 15 min and blocked with 5% bovine serum albumin for 30 min. Then, the cells were incubated overnight at 4°C with anti-GPX4 antibody (1: 200; Abcam; Cambridge, MA, USA), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Amresco) for 1 h at room temperature in the dark. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Subsequently, the stained cells were observed and photographed using a fluorescence microscope (Olympus, Japan).
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