Partial kidney tissues were thoroughly homogenized in a lysis buffer (0.97% protease inhibitor cocktail, 0.94% 50 mM phenylmethylsulfonyl fluoride, and 97.09% 1x RIPA) on ice. The protein concentrations of the homogenates were measured using the BCA Protein Assay Kit (Merck Millipore, USA); 50 μg of protein was electrophoresed on 12% SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA), and blocked in 5% bovine serum albumin (BSA) in Tris-buffered saline. Then, the bands were incubated overnight at 4°C in a corresponding primary antibody solution containing Nrf2 (ab137550), HO-1 (ab13248), SOD1 (ab13498), SOD2 (ab13533), and CAT (ab16731) (1 : 2000; Abcam, UK) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ABS16, 1 : 2000, Merck Millipore, USA) and then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (bs-0295G, 1 : 2000, Beijing Biosynthesis Biotechnology Co. Ltd., China) for 4 hours at 4°C. Specific signals were visualized with ECL detection on a gel imaging system (UVP, California, USA). The average optical density of the bands was quantified using ImageJ (National Institutes of Health, Bethesda, USA).
Ab13498
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab13498 is a laboratory product manufactured by Abcam. It is a component for use in scientific research, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab13533, by Abcam (14 mentions)
Ab16731, by Abcam (9 mentions)
Ab22604, by Abcam (6 mentions)
Ab16801, by Abcam (4 mentions)
Ab13534, by Abcam (4 mentions)
Ab137550, by Abcam (3 mentions)
Ab68155, by Abcam (3 mentions)
Ab8226, by Abcam (3 mentions)
Ab1877, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab31163, by Abcam (2 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (2 mentions)
Ab214430, by Abcam (2 mentions)
Ab62352, by Abcam (2 mentions)
Ab108427, by Abcam (2 mentions)
Ab15323, by Abcam (2 mentions)
Ab86299, by Abcam (2 mentions)
Ab133303, by Abcam (2 mentions)
Ab38898, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
35 protocols using ab13498
1
Protein Expression Analysis in Kidney Tissues
2
Immunoblotting Analysis of Mitochondrial and Antioxidant Proteins
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BCPAP cells or C643 cells were harvested and lysed with RIPA buffer (Millipore). Immunoblotting was performed in a standard fashion. The following antibodies were used: β-actin (1 : 1000; cat #AF5003; Beyotime, China), NDUFB3 (1 : 500; cat #SC-393351; Santa Cruz, CA), MT-ND1 (1 : 1000, cat #ab181848; Abcam), MT-ND2 (1 : 1000, cat #PA5-37185; Invitrogen), MT-ND3 (1 : 1000, cat #ab192306; Abcam), MT-ND4 (1 : 1000, cat #ab219822; Abcam), MT-ND4L (1 : 1000, cat #PA5-103953; Invitrogen), MT-ND5 (1 : 1000, cat #ab230509; Abcam), MT-ND6 (1 : 1000, cat #PA5-109993; Invitrogen), GPX1 (1 : 1000, cat #ab22604; Abcam), MnSOD (1 : 1000, cat #ab68155; Abcam), CuMnSOD (1 : 1000, cat #ab13498; Abcam), PRDX1 (1 : 1000, cat #NBP1-82558; Novus), PRDX3 (1 : 1000, cat #NBP2-67043; Novus), PRDX6 (1 : 1000, cat #H00009588-M01; Novus), and α-tubulin (1 : 1000; cat# AF0001; Beyotime, China).
3
Immunoblotting Protocol for Protein Expression
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After boiling in Laemmli′s sample buffer, proteins (40 μg) were resolved on 7%, 12% or 15% SDS-polyacrylamide gels, and transferred to the PVDF membrane. Membranes were blocked with 5% BSA, and incubated with primary antibody. CTR1, DMT1, ATOX1, COX17, COX2 and CCS were detected using Santa Cruz Biotechnology (Santa Cruz, CA, USA) antibodies: sc-18473, sc-166884, sc-398742, sc-393617, sc-514489, sc-55561, respectively. SOD1, SOD2, CAT, GR and GPX, were detected using Abcam (Trumpington, Cambridge, UK) antibodies: ab13498, ab13533, ab16731, ab16801, ab22604, respectively. Anti-MT-1/MT-2 antibody (M0639) was the product of Dako (Agilent, Santa Clara, CA, USA). β-actin was detected by AC-15 antibody (Sigma-Aldrich, St. Louis, MO, USA). The blots were incubated with anti-rabbit, anti-goat, or anti-mouse secondary antibodies conjugated with horseradish peroxidase. Immunoreactive proteins were visualized by the enhanced chemifluorescence method. Quantitative analysis of immunoreactive bands was done using iBrightTM FL1500 Imaging System Software (Thermo Fisher Scientific, Waltham, MA, USA). All experimental samples and controls used for one comparative analysis are run on the same blot/gel.
4
Western Blot Analysis of SOD1 Protein
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Cells were lysed with a homogenizer in ice-cold Triton X-100 lysis buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1 mM phenylmethanesulfonylfluoride, PMSF) containing Complete Protease Inhibitor Cocktail (Roche, 11697498001). The cell lysates were centrifuged at 12,000 g for 10 min. The supernatants were mixed with sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Then, 20 µg of protein from each sample was separated on a 15% Tris-HCl denaturing gel by SDS-PAGE, transferred to a nitrocellulose membrane for 2 h at 120 V, blocked with 5% bovine serum albumin (BSA), and incubated with primary antibodies for 24 h at 4 °C. The membranes were incubated with HRP-conjugated goat anti-rabbit or mouse secondary antibodies for 2 h at room temperature. Rabbit anti-SOD1 (1:500, Abcam, ab13498) was the primary antibody, while rabbit anti-β-Actin (1:1000, Abcam, ab8227) was used as the internal control.
5
Quantitative Western Blot Analysis
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Western blot analysis was conducted as described previously [26 (link)] using antibodies against: CuZnSOD (0.2 μg ml−1; ab13498), MnSOD (0.2 μg ml−1, ab13533), CAT (1 μg ml−1; ab1877), GSH-Px (1 μg ml−1; ab17926-500), Nrf2 (1 μg ml−1; ab31163), 4-HNE (1 μg ml−1; ab46545) and β-actin (0.5 μg ml−1; ab8226) (all purchased from Abcam, Cambridge, UK). Quantitative analysis of immunoreactive bands was performed with ImageJ software (National Institute of Health, USA). Total band density was calculated as the sum of pixel intensities within a band. The ratio of dots per band for the target protein was averaged against β-actin (gel loading control) from three independent experiments, and the levels of protein expression were expressed in arbitrary units (AU).
6
Immunoblot Analysis of Apoptosis Markers
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The hepatocytes were lysed with ice-cold RIPA buffer and the protein extraction was immunoblotted to analyze the protein expression. The protein lysates were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Merck Millipore Co, Ltd. USA). Immunoblotting was performed according to the standard procedures with the following primary antibodies: β-actin (4,970, 1:1,000), BAX (5,023, 1:1,000), BCL-2 (2,870, 1:1,000), Caspase-3 (9,665,1:1,000), SOD2 (13,141,1:1,000), and Cytochrome C (11,940, 1:1,000) antibodies [Cell Signaling Technology (Beverly, MA, USA)], antibodies for SOD1 (ab13,498,1:5,000) and Caspase-9 (ab219590, 1:1,000) [obtained from Abcam (Cambridge, MA, USA)], and goat anti-rabbit IgG (H + L) HRP (BS13278, 1:5,000) [obtained from Bioworld Technology (St. Paul, MN, USA)]. The membranes were exposed and visualized using the ECL immobilon western chemiluminescent HRP substrate (WBKLS0500, Millipore, USA). Quantitative analysis was performed using Quantity One software (Bio-Rad Laboratories).
7
Tanshinone IIA Protects C3H10T1/2 Cells from Oxidative Stress
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C3H10T1/2 cells provided by the National Collection of Authenticated Cultures, Chinese Academy of Sciences were plated in a 6-well plate at a density of 8 × 104 cells/well for 24 h and were pretreated with 0, 10− 8, 10− 7, or 10− 6M Tanshinone IIA for 24 h. After incubation in 500µM H2O2 for another 24 h, the cells were collected, and total proteins were extracted on ice using radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China). The protein concentration was detected using an enhanced bicinchoninic acid assay kit (P0010, Beyotime Biotechnology, Shanghai, China). Western blot analysis was conducted as previously described [18 ]. The following antibodies were used: anti-cleaved caspase 3 (ab214430), anti-pro caspase 3 (ab32499), anti-superoxide dismutase 1 (SOD 1, ab13498), anti-heme oxygenase-1 (HO-1, ab68477), anti-catalase (CAT, ab16731) from Abcam (Cambridge, UK) and anti-Nrf2 (12721), anti-β-actin (8457), anti-Keap1 (8047), anti-Bcl2 (3498) and anti-Bax (5023) from Cell Signaling Technology (Danvers, Massachusetts, USA). A chemiluminescent horseradish peroxidase substrate kit (WBKLS0500, Millipore, Billerica, Massachusetts, USA) was used for the electrochemiluminescence detection assay.
8
Protein isolation and Western blot analysis
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Total protein was obtained from frozen heart tissue and H9c2 cells, the cytosolic and nuclear proteins were isolated using a commercial kit (78833, Thermo Fisher Scientific, United States) as described in our previous study (Hu et al., 2021b (link)). Then, total protein was loaded into 10% SDS-PAGE gels, electro-transferred to an Immobilon-P membrane (IPVH00010, Millipore, China) and incubated with the primary antibodies against cTnI (ab52862, Abcam, United States), p-cTnI (ab58546, Abcam, United States), SOD1 (ab13498, Abcam, United States), SOD2 (ab13533, Abcam, United States), Sirt1 (ab189494, Abcam, United States), p-Nrf2 (ab76026, Abcam, United States), Nrf2 (ab62352, Abcam, United States), heme oxygenase-1 (HO-1, ab68477, Abcam, United States), Lamin B1 (ab16048, Abcam, United States) and GAPDH (ab8245, Abcam, United States). After incubation with the secondary antibody, the membranes were scanned using an Odyssey infrared imaging system (LI-COR, United States), and GAPDH was selected as the internal control.
9
Western Blot Analysis of Antioxidant Enzymes
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Whole cell extracts from control and treated uteri were mixed 1 : 1 with Laemmli sample buffer, boiled for 5 min, and stored at −80°C until further analysis. Proteins were resolved in 12% SDS polyacrylamide gels and transferred to PVDF membranes. SOD1, SOD2, CAT, GSH-Px, and GR were detected by Abcam antibodies (ab13498, ab13533, ab16731, ab22604, and ab16801, resp.). β-Actin was detected by AC-15 antibody (Sigma-Aldrich). After incubation with secondary antibodies, the immunoreactive proteins were visualized by the enhanced chemifluorescence (ECF) method (Amersham Biosciences Limited, UK). Quantitative analysis of immunoreactive bands was performed using the ImageQuant software. β-Actin was used as equal load control.
10
HepG2 Exposure to Lp(a) and LDL
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Human hepatocellular carcinoma (HepG2) cells (American Type Culture Collection, Manassas, VA) were maintained in Advanced Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Bio International, Auckland, New Zealand), 2 mM L-glutamine, 0.25 μg/mL amphotericin B, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies) at 37°C in a humidified environment with 5% CO2. HepG2 cells were seeded at 5×105 cells/mL. 24 hours after seeding, cells were treated with 5 μg/ml of either purified Lp(a) or LDL for 6 hours at 37°C. Cell lysates were harvested and 40 μg of cell lysate protein was subject to western blot analysis with anti-Gpx1 (ab108427, Abcam), anti-Prdx6 (ab16947 Abcam), and anti-SOD (ab13498 Abcam) antibodies. An anti-actin antibody (Novus NB100-74340) was used as a loading control. Protein levels were quantified by densitometry using Image Studio Lite (LI-COR Biosciences, Inc) after normalisation to actin.
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