Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis. MO, USA). MES23.5 cells were provided by Professor Wei-Dong Le (Baylor College of Medicine, TX, USA). Nesfatin-1 was obtained from Phoenix Pharmaceuticals Inc. (Burlingame, CA, USA). Dulbecco’s modified Eagle’s medium nutrient mixture-F12 (DMEM/F12) was purchased from Gibco (Grand Island, NY, USA). The PE-conjugated monoclonal active-caspase-3 antibody apoptosis kit was from BD Bioscience (San Diego, CA, USA). PD98059 and the BCA kit was from Beyotime (Shanghai, CN). KT5720 and anti-rabbit secondary antibody conjugated to horseradish peroxidase were from Santa Cruz (Santa Cruz, CA). The cell fractionation kit was from Clontech (Mountain View, CA, USA). Anti-Cox IV monoclonal antibody was from Clontech (CA, USA). The primary antibody against TH was from Millipore (Darmstadt, Germany). Primary antibodies against ERK1/2 and phospho-ERK1/2 were from cell signaling technology (MA, USA). Alexa Fluor 555 donkey anti-rabbit secondary antibody was from Eugene (OR, USA). GW5074 was from Selleck (TX, USA).
Anti rabbit secondary antibody conjugated to horseradish peroxidase
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-rabbit secondary antibody conjugated to horseradish peroxidase is a laboratory reagent used in various immunoassay techniques. It binds to rabbit primary antibodies and enables detection through the enzymatic activity of the horseradish peroxidase conjugate.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay kit, by Bio-Rad (3 mentions)
Dulbecco s modified eagle s medium nutrient mixture f 12 dmem f 12, by Thermo Fisher Scientific (1 mentions)
Bca kit, by Beyotime (1 mentions)
Kt5720, by Santa Cruz Biotechnology (1 mentions)
Gw5074, by Selleck Chemicals (1 mentions)
Pe conjugated monoclonal active caspase 3 antibody apoptosis kit, by BD (1 mentions)
Primary antibodies against erk1 2 and phospho erk1 2, by Cell Signaling Technology (1 mentions)
Nesfatin 1, by Phoenix Pharmaceuticals (1 mentions)
Pd98059, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Biodoc it imaging system, by Analytik Jena (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
F4 80, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Biodoc imaging system, by Analytik Jena (1 mentions)
6 protocols using anti rabbit secondary antibody conjugated to horseradish peroxidase
1
Nesfatin-1 Induced Apoptosis Pathway
2
Lactoferrin Modulates Oxidative Stress in Neurodegeneration
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After pretreatment with 100 ng/ml apo-Lf or 100 ng/ml holo-Lf, VM neurons were incubated with 100 μmol/L MPP+ for 24 h. To examine the expressions of LfR, Cu/Zn-SOD, Bcl-2 and Bax, cells were digested with RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF) with protease inhibitors (pepstatin 1 μg/mL, aprotinin 1 μg/mL, leupeptin 1 μg/mL) for 30 min. The lysate was centrifuged at 12,000 g for 20 min at 4 °C, and the supernatant was used for analysis. Protein concentration was established using the BCA protein assay kit (Beyotime, Jiangsu, China). A total of 25 μg of protein from each sample was separated using 10% SDS-polyacrylamide gels and transferred to PVDF membranes. After 2 h blocking with 10% non-fat milk at room temperature, the membranes were incubated with rabbit-anti-rat LfR (1:6000), Cu/Zn-SOD (1:10000), Bcl-2 (1:1000), Bax (1:1000) antibodies over night at 4 °C. Anti-rabbit secondary antibody conjugated to horseradish peroxidase was used at 1:10000 (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were also probed with anti-β-actin antibody (1:10000) as a loading control. Cross-reactivity was visualized using ECL Western blotting detection reagents and analyzed by scanning densitometry using a UVP Image System (Upland, CA, USA).
3
Western Blot Analysis of Oxidative Stress Markers
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VM neurons or astrocytes were treated as described above, washed with cold PBS, and lysed with lysis buffer containing 50 mmol/L Tris HCl, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (pepstatin 1 μg/mL, aprotinin 1 μg/mL, leupeptin 1 μg/mL) for 30 min on ice, and the insoluble material was removed by centrifugation (12,000 rpm, 20 min, 4 °C). Protein concentration was determined by the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA). A total of thirty micrograms of protein was separated using 10% SDS-polyacrylamide gels and transferred to NC membranes. After blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated with rabbit anti-rat primary antibodies against HO-1 (1:2000) or MtFt (1:1000) for 2 h at 4 °C. Anti-rabbit secondary antibody conjugated to horseradish peroxidase was used at 1:10,000 (Santa Cruz Biotechnology, Santa Cruz, CA). β-actin and COX4 were detected using mouse anti-β-actin monoclonal antibody (1:8000) according to a similar procedure that ensured equal samples of total protein and mitochondrial protein, respectively. Cross-reactivity was visualized using ECL Western blot detection reagents and analyzed via scanning densitometry using a UVP BioDoc-It Imaging System (UVP, Upland, USA).
4
Western Blot Analysis of F4/80 Protein
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Forty micrograms of protein was separated on 7.5-10% SDS/PAGE and transferred onto nitrocellulose membranes. After blocking for 1 h in 5% skim milk solution, the membranes were incubated with specific antibodies to F4/80 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were then exposed to an anti-rabbit secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The signals were detected by chemiluminescence using the ECL detection reagent (GE Healthcare, Piscataway, NJ, USA). The bands were scanned with a ChemiDoc XRS System (Bio-Rad Laboratories) with a cooled 12-bit camera and quantified by densitometry. Levels of target proteins were normalized to β-actin values.
5
Western blot analysis of iron-related proteins
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BV2 microglial cells were treated as described above. Following three washes with cold phosphate-buffered saline, cells were lysed with lysis buffer containing 50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors (pepstatin 1 μg/mL, aprotinin 1 μg/mL, leupeptin 1 μg/mL) for 30 min on ice. The insoluble pellet was removed by centrifugation (12,000 rpm, 20 min, 4 °C) from each sample. Protein concentration was determined by the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA). A total of 30 μg of protein was resolved by running in 8% SDS-polyacrylamide gels and was subsequently transferred to PVDF membranes. After blocking with 10% non-fat milk at room temperature for 2 h, the membranes were incubated with rabbit anti-rat IRP1 (1:800), DMT1 (1:800), and FPN1 (1:800) antibodies overnight at 4 °C. Anti-rabbit secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a 1:10,000 dilution. Blots were also probed with anti-β-actin antibody (1:10,000) as a loading control. Cross-reactivity was visualized using ECL western blotting detection reagents and analyzed by scanning densitometry using a UVP BioDoc-Imaging System (UVP, Upland, CA, USA).
6
Western Blot Analysis of Hypoxia-Responsive Proteins
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Astrocytes and VM neurons were treated as described above and after three washes with cold phosphatebuffered saline, cells were lysed with lysis buffer containing 50 mM Tris HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl uoride (PMSF), and protease inhibitors (pepstatin 1 μg/ml, aprotinin 1 μg/ml, leupeptin 1 μg/ml) for 30 min on ice. The insoluble material was removed by centrifugation (12000 rpm, 20 min, 4°C). Protein concentration was determined by the Bradford assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). A total of 30 μg of protein was separated using 8% SDS-polyacrylamide gels and transferred to Polyvinylidene Fluoride (PVDF) membranes. After blocking with 10% non-fat milk at room temperature for 2 h, the membranes were incubated with rabbit anti-rat HIF-1α (1:1000), or HIF-2α (1:1000), or DMT1 (1:800), or FPN1 (1:800) or PKCδ (1:800) and p-PKCδ (1:800) antibodies over night at 4°C. Anti-rabbit secondary antibody conjugated to horseradish peroxidase was used at 1:10000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were probed with anti-β-actin antibody (1:10000) as a loading control. Cross-reactivity was visualized using ECL Western blotting detection reagents and analyzed by scanning densitometry using a UVP BioDoc-Imaging System (UVP, Upland, CA, USA).
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