Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was washed three times and blocked by soaking in Tris-buffered saline + Tween (TBS-T) buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) containing 5% bovine serum albumin (GenDEPOT) for 30 min. The membrane was incubated with primary antibodies (1:1000) overnight at 4 °C. Then, the membrane was washed three times and incubated with the appropriate secondary antibody (1:2000) for 1 h at room temperature. All primary and secondary antibodies (β-actin, Nrf2, HO-1, SOD2, catalase, Gpx-1, Bax, Bcl/Bcl-xL, JNK/p-JNK, ERK/p-ERK, and p38/p-p38) were obtained from Abcam (Cambridge, UK) and Cell Signaling Technology (Beverly, MA, USA). Protein expression was visualized using enhanced chemiluminescence reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Quantitative analysis was conducted using ImageJ software (version 1.52a for Windows; NIH, Bethesda, MD, USA).
Catalase
Manufactured by Abcam
Sourced in United States, United Kingdom
Catalase is an enzyme that catalyzes the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2). It is an essential enzyme found in most living organisms that are exposed to oxygen, as it helps to protect cells from the potentially damaging effects of hydrogen peroxide.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (6 mentions)
β actin, by Abcam (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Mnsod, by Merck Group (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Bcl2 associated x (bax), by Abcam (3 mentions)
α tubulin, by Merck Group (3 mentions)
Ab1877, by Abcam (3 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Imagequant las 4000, by GE Healthcare (3 mentions)
Total oxphos, by Abcam (3 mentions)
Bcl 2, by Abcam (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Superoxide dismutase 1, by Abcam (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Foxo3, by Cell Signaling Technology (2 mentions)
P yap, by Cell Signaling Technology (2 mentions)
49 protocols using catalase
1
Western Blot Analysis of Protein Expression
2
Chaetocin Induces Melanoma Cell Apoptosis
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Human melanoma cell lines, Sk-Mel-28, A375, IGR37, LU-1205 and MV3 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and they were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), penicillin (100 IU/ml), and streptomycin (100 μg/ml) in a humidified incubator with 5% CO2 at 37°C. Human primary melanocytes were obtained Kanglang Company (Shanghai, China) and cultivated in melanocytes growth with 10% FBS. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) and chaetocin were purchased from Sigma-Aldrich Corp. (St. Louis, MO). chaetocin was dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution which was diluted to the final concentration with culture medium. The final concentration of DMSO was kept under 0.1% throughout the following studies, and showed no effect on cell morphology and proliferation in this study. The primary antibodies recognizing Bax, Bcl-2, procaspase-9/-3, cleaved caspase-9/-3, cytochrome c, PCNA, Nrf2, SOD2, catalase, and β-actin were purchased from Abcam Company (Cambridge, UK).
3
Protein Expression Analysis in Cells
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After the indicated treatments, cells were lysed by ice-cold RIPA buffer containing proteasome-inhibitor cocktail (Cell Signaling, Danvers, MA). Protein samples were separated by SDS-PAGE, transferred onto a polyvinylidene difluride membrane (Thermo Scientific, Rockford, IL), and probed with an appropriate antibody. Antibodies against caspase-7, caspase-9, p53 were purchased from BD Pharmingen (San Diego, CA). Anti- human RIP1, RIP3, GPX1, Catalase, γ-H2AX, H2AX, and PGAM5 antibodies were from Abcam Inc. (Cambridge, MA). Anti-LC3 antibody was purchased from Novus Biological Inc. (Littleton, CO). TrxR1 antibody (B-2) and glutathione synthetase (GSS) antibody (C-5) were from Santa Cruz Biotechnology (Santa Cruz, CA). A total of 30 μg protein was used for the immunoblotting, unless otherwise indicated. GAPDH was used for the loading control.
4
Apoptosis Detection and Analysis
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Annexin V-FITC Apoptosis Detection Kit, Phalloidin–Tetramethylrhodamine B isothiocyanate and antibody to β-actin were purchased from Sigma (USA). CMH2DCFDA and TMRM from Life Technology, (USA), Suicide Track™ DNA Ladder Isolation Kit from Calbiochem (USA), RapiGest SF Surfactant from Waters (UK). Antibodies Chop, Hsp70, caspase3, caspase7, caspase9, catalase and PARP was purchased from Cell signaling technology (USA). catalase and Peroxiredoxin3 were purchased from Abcam USA and secondary antibodies (HRP-conjugated anti-mouse and anti-rabbit were purchased from Santa Cruz biotechnology (USA).
5
Western Blot Analysis of Sciatic Nerve
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Sciatic nerve proteins from each group were separated on 12% polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. These membranes were incubated for 2 hours at room temperature in 3% bovine serum albumin. The primary polyclonal antibodies were rabbit anti-rat aldose reductase (37 kDa), glutathione peroxidase 1 (GPX, 22 kDa), superoxide dismutase (SOD, 18 kDa), catalase (CAT, 60 kDa) (1:1,000, 1:5,000, 1:1,000, 1:2,000; Abcam, Cambridge, MA, USA). The membranes were incubated with primary antibodies overnight at 4°C and with secondary antibody (goat anti-rabbit IgG, monoclonal antibody; 1:15,000; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) over 2 hours at room temperature. After three washes in Tris-buffered saline with Tween, immunoreactive complexes were visualized using an enhanced chemiluminescence system (Bio-Rad, Hercules, CA, USA). β-Actin served as an internal positive control.
6
Quantification of Antioxidant Enzymes
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The small mesenteric arteries and small renal vessels were placed into ice-cold homogenization buffer (same composition as that in the previous sec tion) containing protease inhibitors (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitors (Pierce, Pockford, IL, USA), and homogenized on ice with a glass tissue grinder. Protein concentration was determined by standard Bradford assay and western blotting, as described previously (23 (link)). Briefly, after transferring the proteins onto a PVDF membrane, the membrane was blocked in a blocking buffer (LI-COR Biosciences, Lincoln, NE, USA). Two-color immunoblotting was performed using primary antibodies against SOD1 (Stressgen, Victoria, British Columbia Canada), SOD2 (Stressgen, Victoria, British Columbia Canada), SOD3 (generously provided by Dr. Tohru Fukai, Augusta University), catalase (Abcam, Cambridge, MA, USA), and β-actin (Sigma-Aldrich). Specific bands were detected using an Odyssey Infrared Imager (LI-COR Biosciences, Lincoln, NE, USA). IRDye800 (Rockland Immunochemicals, Limerick, PA, USA) and AlexaFluor 680 (Molecular Probes, Eugene, OR, USA) were used as secondary antibodies to detect two primary antibodies from different species. Relative densitometric units of the proteins were calculated via normalization to that of β-actin.
7
Pisum sativum Antioxidant Enzyme Assay
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The voucher specimens (Pisum sativum L. var. saccharatum Poir: CMU-104-PS-003) were deposited in Herbarium of College of Pharmacy, China Medical University, Taichung, Taiwan. Antipain, aprotinin, dithiothreitol, ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, leupeptin, Nonidet P-40, pepstatin, phenylmethylsulfonyl fluoride, sodium deoxycholate, trigonelline, and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) were purchased from Sigma Chemical Company (St. Louis, MO). Antibodies to various proteins were obtained from the following sources: β-actin antibody was purchased from Sigma Chemical Company; Caspase-9, catalase, p38 (pThr180/Tyr182), and Raf (pSer259) were purchased from Abcam (Cambridge, MA); Mn-SOD and Cu/Zn-SOD were from Calbiochem (San Diego, CA); ERK (pThr202/Tyr204) was from ThermoFisher Scientific, Inc. (Waltham, MA); Nrf2 (pSer40) was from GeneTex, Inc. (Irvine, CA); Caspase-3 and protein kinase Cα (PKCα) from BD Biosciences (San Jose, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and -rabbit IgG were from Abcam.
8
Western Blot Analysis of Cellular Proteins
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The proteins were isolated from MEFs as described previously [87 (link)]. Proteins at a concentration of 20 μg/μL were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane (Roche, Basel, Switzerland). After blocking, the membranes were incubated overnight at 4 °C with the following primary antibodies: Hif-1α, Cell Signaling Technology D2U3T #14179; catalase, Abcam ab16731; superoxide-dismutase 1, Abcam ab16831; fatty acid synthase, Santa Cruz sc-48357; trifunctional enzyme subunit beta, mitochondrial, Hadhb, Santa Cruz sc-271496; phospho-AMPK (Thr172), phosphorylated AMP-induced protein kinase, Cell Signaling (40H9). An appropriate horseradish peroxidase (HRP)-conjugated secondary antibody was utilized for chemiluminescence detection. Total protein normalization was achieved using AmidoBlack (Sigma Aldrich, St. Louis, MO, USA). Immunoblots were detected using the Alliance 4.7 Imaging System (UVITEC, Cambridge, UK) and an enhanced chemiluminescence kit (Thermo Fischer Scientific, Waltham, MA, USA). Experiments for each protein of interest were performed at least two times.
9
Immunoblotting Antibody Specifications
10
Western Blot Analysis of Catalase and GAPDH
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Extracted proteins (10 μg) were separated using SDS-polyacrylamide gel electrophoresis under reducing and denaturing conditions and transferred to a 0.45 μm polyvinylidene difluoride membrane. The membranes were blocked with tris-buffered saline (TBS)-Tween containing 10% (w/v) skimmed milk powder before incubation overnight (4°C) with a primary antibody diluted in TBS-Tween containing 5% (w/v) BSA. Primary antibodies used included catalase (1:2000, Abcam) and GAPDH (1:10,000, Cell Signaling). Membranes were then incubated with an anti-rabbit horseradish peroxidase conjugated secondary antibody (1:10,000, GE Healthcare Life Sciences) and proteins were visualized using the enhanced chemiluminescence system (Amersham). Protein bands were quantified by densitometry with ImageJ 1.46r software.
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