Antibodies for β-actin (sc-47778), COX2 (sc-19999), MMP1 (sc-21731), NRF1 (sc-101102), and SOD2 (sc-30080) were obtained from Santa Cruz Biotechnology. Antibodies for acetyl-histone H4 K5, K8, K12, and K16 (H4K5,8,12,16ac, #PA5-40084) were obtained from Invitrogen. Antibodies for anti-histone H3 acetyl K9, K14, K18, K23, K27 (H3K9, 14, 18, 23, 27ac, ab47915), ERα (ab3575), ERβ (ab3576), H4K20me1 (ab9051), H4K20me3 (ab9053), H4R3me1 (ab17339), H3K9me2 (ab1220), H3K9me3 (ab8898), H3K27me2 (ab24684) and H3K27me3 (ab6002), H2AX (ab20669), and γH2AX (ab2893) were obtained from Abcam. The antibody for 8-oxo-dG (4354-MC-050) was obtained from Novus Biologicals. The 3-nitrotyrosine (3-NT) was measured using 3-Nitrotyrosine ELISA Kit (ab116691 from Abcam) per manufacturers’ instructions. The mitochondrial fraction was isolated using a Pierce Mitochondria Isolation Kit (Pierce Biotechnology) according to manufacturers’ instructions. The protein concentration was measured using the Coomassie Protein Assay Kit (Pierce Biotechnology) per manufacturers’ instructions. Luciferase activity assay was carried out using the Dual-Luciferase™ Assay System (Promega) and the transfection efficiency was normalized using a cotransfected renilla plasmid (23 (link)). 17β-estradiol (E2, #E2758) and TNFα (#T0157) were obtained from Sigma.
Sc 30080
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-30080 is a lab equipment product from Santa Cruz Biotechnology. It serves as a tool for researchers in their scientific investigations, but a detailed and unbiased description of its core function cannot be provided while maintaining the requested parameters.
Automatically generated - may contain errors
Lab products found in correlation
Sc 11407, by Santa Cruz Biotechnology (4 mentions)
Ab6002, by Abcam (2 mentions)
Coomassie protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pierce mitochondria isolation kit, by Thermo Fisher Scientific (2 mentions)
Ab7778, by Abcam (2 mentions)
Enhanced chemiluminescence detection system, by Santa Cruz Biotechnology (2 mentions)
Sc 25778, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab3576, by Abcam (1 mentions)
Ab9051, by Abcam (1 mentions)
Ab17339, by Abcam (1 mentions)
Ab24684, by Abcam (1 mentions)
Ab9053, by Abcam (1 mentions)
3 nitrotyrosine elisa kit, by Abcam (1 mentions)
17β estradiol e2 e2758, by Merck Group (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Ab37438, by Abcam (1 mentions)
Ab20669, by Abcam (1 mentions)
Ab8898, by Abcam (1 mentions)
Ab1220, by Abcam (1 mentions)
12 protocols using sc 30080
1
Antibody-based Protein Quantification in Cellular Studies
2
Mitochondrial and Nuclear Protein Extraction
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The antibodies for ERRα (ab37438), H2AX (ab20669), γH2AX (ab2893), H3K9me2 (ab1220), H3K9me3 (ab8898) and H3K27me3 (ab6002) were obtained from Abcam, and all the other antibodies, including β-actin (sc-47778), eNOS (sc-654), ERβ (sc-137381) and SOD2 (sc-30080) were obtained from Santa Cruz Biotechnology. The mitochondrial fraction was isolated using a Pierce Mitochondria Isolation Kit (Pierce Biotechnology) according to manufacturers’ instructions. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotechnology). Protein concentration was measured using the Coomassie Protein Assay Kit (Pierce Biotechnology) according to manufacturers’ instructions.
3
Western Blot Analysis of Protein Expression
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HOBs were lysed in freshly prepared ice-cold RIPA buffer. 35 µg total protein, quantified by micro Lowry, was separated by SDS page and transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in TBS-T for 1 h at ambient temperature followed by overnight incubation at + 4 °C with primary antibodies (sc-32886, sc-30147, sc-50508 and sc-30080 Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000 in TBS-T. The next day membranes were incubated with the corresponding peroxidase-labeled secondary antibodies (1:10,000 in TBS-T) for 2 h at ambient temperature. GAPDH was used as a loading control. For signal development membranes were incubated for 1 min with ECL substrate solution. Chemiluminescent signals, detected by a CCD camera (INTAS, Göttingen, Germany), were quantified using the ImageJ software.
4
Diabetic Cardiac Fibrosis Evaluation
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THC was purchased from Shanghai Macklin Biochemical Co. Ltd. (Shanghai, China) with a purity of 98% tested by high-performance liquid chromatography (HPLC). STZ was purchased from Sigma-Aldrich (St. Louis, MO, USA). The fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) used to detect intracellular ROS generation was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Dihydroethidium (DHE; for detecting ROS generation in cardiac tissues) were purchased from Invitrogen (Carlsbad, CA, USA). A primary antibody against SOD2 (sc-30080) was obtained from Santa Cruz Biotechnology (CA, USA). Primary antibodies against T-Smad3 (SC101154), p-Smad3 (8760), and β-actin (4970) were all purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibodies against SIRT1 (ab110304), acetylated SOD2 (Ac-SOD2, ab137037), α-SMA (ab5694), TGFβ1 (ab92486), collagen І (ab90395), and collagen III (ab7778) were purchased from Abcam (Cambridge, MA, USA). The rabbit anti-goat and goat anti-mouse secondary antibodies were obtained from the Zhongshan Company (Beijing, China).
5
Nrf2-Keap1 Pathway Regulation Analysis
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Whole-cell and nuclear extracts were prepared as previously described [35 (link)]. Western blot analysis was performed as described previously [29 (link)]. Briefly, cell extracts (10–40 μg/lane) were separated by 8–10% SDS polyacrylamide gel electrophoresis and electroblotting, and proteins were detected using antibodies against p-Nrf2 (ab76026, Abcam), Nrf2 (ab62352, Abcam), Keap1 (sc-365626, Santa Cruz Biotechnology), HO-1 (ADI-SPA-895-F, Enzo Life Sciences, NY, USA), SOD-1 (sc-11407, Santa Cruz Biotechnology), SOD-2 (sc-30080, Santa Cruz Biotechnology), lamin B1 (ab16048, Abcam), and β-actin (sc-47778, Santa Cruz Biotechnology). After detection with horseradish peroxidase-conjugated secondary antibody (anti-mouse or -rabbit), proteins were visualized using an enhanced chemiluminescence detection system (Santa Cruz Biotechnology) and an EZ-Capture ST imaging system (Atto, Tokyo, Japan). β-actin was used as the loading control. The densitometry data represent the mean ± standard error (SE) from three immunoblots and are shown as the relative density of protein bands normalized to the indicated protein (input Nrf2, input Keap1, actin, or lamin B1).
6
Protein Extraction and Western Blot Analysis
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Protein was extracted from the heart tissues and cells using a protein extraction kit (BC3710; Beijing Solarbio Science & Technology Co., Ltd Solarbio, Beijing, China) and protein concentration was quantified using a BCA assay (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. Protein (20 µg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (EMD Millipore). Following blocking with 5% nonfat milk in Tris buffered saline Tween-20, the blots were incubated with primary antibodies: Anti-NOD2 (1:500; sc-30080; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-collagen I (1:1,000; ab34710; Abcam, Cambridge, MA, USA), anti-collagen III (1:1,000; ab7778; Abcam), anti-TGF-β (1:1,000; ab31013; Abcam) and GAPDH (1:5,000; sc-25778; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase-conjugated rabbit secondary antibodies (1:2,000; K5007; Dako, Agilent Technologies, Inc., Santa Clara, CA, USA) for 1 h at room temperature. The protein bands were visualized using the SuperSignal chemiluminescent detection module (34080; Pierce, Thermo Fisher Scientific, Inc.) and images were collected using a Tanon-5200 imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China).
7
Protein Expression Analysis of ELIT Treatment
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After 48 h of ELIT treatment, cells were harvested as described previously by Torrens-Mas [35 (link)]. Protein content (supernatant) was determined with the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Ten micrograms of protein were resolved on a 12% SDS-PAGE gel and electrotransferred onto nitrocellulose membranes using the Trans-blot® Turbo™ transfer system (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% non-fat powdered milk in TBS with 0.05% Tween for 1 h. Antisera against OXPHOS complexes (ab110411; Abcam, Bristol, UK), SOD-1 (574597, Calbiochem®, San Diego, CA, USA), SOD-2 (sc-30080; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CAT (219010, Calbiochem®, San Diego, CA, USA), GRd (sc-133245; Santa Cruz Biotechnology, Santa Cruz, CA, USA), 4-HNE (HNE11-S, Alpha Diagnostic, San Antonio, TX, USA), COXIV (ab33985, Abcam, Bristol, UK), PGC1α (ab54481, Abcam, Bristol, UK), and GAPDH (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as primary antibodies. Protein bands were visualized as described previously by [34 (link)].
8
Antibody Staining for Key Proteins
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The antibodies for β-actin (sc-47778), CYP19 (sc-374176), RORA (sc-518081) and SOD2 (sc-30080) were obtained from Santa Cruz Biotechnology. Fluorescein isothiocyanate-labeled dextran (FITC-dextran, #46944) and RORA agonist SR1078 (#557352) were obtained from Sigma.
9
Quantifying Oxidative Stress Markers in Bone Tissue
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Immunohistochemistry staining was performed using anti-SOD1 and SOD2 antibodies to detect OS levels. Briefly, mice were sacrificed 5 weeks after tumor implantation, and right tibia samples were fixed by 4% PFA for two days and then decalcified thoroughly by daily change of buffer, 10% EDTA w/v in water (pH = 7.5), for three weeks. Tibia paraffin sections were antigen unmasked by using sodium citrate buffer (pH = 6.0) and treated with normal serum to block nonspecific binding. Tibia bone tissue sections were labeled with anti-SOD1 polyclonal antibody (1:100 dilution, sc-11407, Santa Cruz, Dallas, TX, USA) and anti-SOD2 polyclonal antibody (1:150 dilution, sc-30080, Santa Cruz) at 4 °C overnight. The sections were then followed by biotin-labeled secondary antibody and signal amplification, DAB kit (Vector, Burlingame, CA, USA) according to manufacturer’s protocols. Hematoxylin was applied for nuclear counterstain. The stained tissue sections were photographed, and the percentage of SOD1- and SOD2-positive cells was quantified using NIH ImageJ software.
10
Mitochondrial Protein Immunoblotting
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Subsequently, membranes were blocked using Li-COR Blocking Buffer (Li-COR, Lincoln, NE) for 1 hour at room temp. Mitochondrial proteins were probed for with the following primary antibodies: 3-mercaptopyruvate sulfurtransferase (3MPST, HPA001240, Sigma), mitochondrial heat shock protein 70 (mtHSP70, ab6535, Abcam), GrpE protein homolog 1 (Grpel1, sc-242966, Santa Cruz), Sirtuin 3 (SIRT3, ab86671, Cell Signaling), superoxide dismutase 2 (SOD2, sc-30080, Santa Cruz), and glutathione reductase (GR, ab16801, Abcam).Soluble proteins were probed for with the following primary antibodies: heat shock protein 70 (HSP70, ab6535, Abcam) and superoxide dismutase 1 (SOD1) (sc-11407, Santa Cruz). All primary antibodies were diluted in Li-COR Blocking Buffer at a 1:1000 ratio, with the exception of Grpel1 (1:200 ratio). Incubations were performed overnight at 4°C. Then membranes were washed in TBS-T (3 ˣ 5 min), and incubated in secondary antibody (IR Dye, 1:20,000, LI-COR) for 30 min at room temp, washed again (TBS-T, 3 ˣ 5 min), and rinsed in 1 ˣ TBS. Finally, membranes were scanned using an Odyssey Infrared Imaging System (LI-COR). For mitochondrial proteins, the immunoblot signal of each target protein was normalized to cytochrome c signals (Cyt c, sc-13156, Santa Cruz) and soluble proteins were normalized to ɑ-tubulin signals (12G10, DSHB).
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