For protein extraction, cells were lysed in cell lysis buffer containing 140 mM NaCl, 10 mM Tris-HCl, 1% Triton X-100, 1 mM ECTA and 1X protease inhibitor cocktail (Gibco; Thermo Fisher Scientific, Inc.). The protein concentration was determined using the BCA assay. A total of 20 µg protein was loaded per lane and separated on 12% SDS-PAGE gels. Proteins were subsequently transferred to polyvinylidene difluoride membranes, which were blocked with 5% non-fat dry milk for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-GAPDH (cat no. sc-25778; 1:2,000) anti-BMP6 (cat no. sc-7406; 1:100; both Santa Cruz Biotechnology, Inc.) and anti-VEGF (cat no. ab105219; 1:1,000; Abcam). They were subsequently incubated with an anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (cat no. sc-2004, 1:1,000, Santa Cruz Biotechnology, Inc.) at 4°C for overnight, and detected with an enhanced chemiluminescence detection kit (EMD Millipore, Billerica, MA, USA).
Anti goat horseradish peroxidase hrp conjugated secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody is a laboratory reagent used to detect and quantify the presence of goat primary antibodies in immunoassays. The HRP enzyme catalyzes a colorimetric reaction, allowing for the visualization and measurement of the target antibody.
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Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence detection kit, by Merck Group (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti vegf, by Abcam (1 mentions)
Albumin from chicken egg white, by Merck Group (1 mentions)
Signalboost immunoreaction enhancer kit, by Merck Group (1 mentions)
Anti ace2 antibody, by R&D Systems (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ykl 40, by Cell Signaling Technology (1 mentions)
Ykl 40, by Abcam (1 mentions)
Akr1c1, by Abcam (1 mentions)
Akr1c3, by Abcam (1 mentions)
Txnrd1, by Abcam (1 mentions)
Anti rabbit and anti mouse hrp conjugated secondary antibodies, by Cell Signaling Technology (1 mentions)
L glutamine, by Nacalai Tesque (1 mentions)
Mem nonessential amino acid solution, by Nacalai Tesque (1 mentions)
5 protocols using anti goat horseradish peroxidase hrp conjugated secondary antibody
1
Protein Extraction and Western Blot Analysis
2
Quantitative Western Blotting of ACE2
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Western blotting assay was performed as previously described (Yang et al., 2011 (link)). Protein extractions from cell lysates were obtained with RIPA buffer mixed protease inhibitor cocktail (Thermo) according to the manufacturer's instructions. Protein extractions were boiled in SDS-PAGE loading buffer for 5 min, and resolved on a SDS-PAGE, then transferred onto PVDF membrane. Membranes were blocked with 2% albumin from chicken egg white (Sigma) for 1 h, and incubated with anti-ACE2 antibodies (R&D) and anti-β-actin antibodies (Sigma) overnight at 4 °C. After triple wash with TBST, they were incubated with anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz) for 1 h at room temperature. Final detection of protein was performed using the Signal Boost Immunoreaction Enhancer Kit (Merck Millipore). Protein levels were quantified by using Quantity One software (Bio-Rad).
3
Protein Extraction and Immunoblotting Protocol
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Cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitor cocktail). The total protein concentration was determined by a BCA protein assay kit (Thermo Fisher, #23225). Cell lysates were mixed with 5× Laemmli sample buffer, boiled for 4 min at 95°C, and then stored at −20°C. Total cell extracts were resolved on an SDS–PAGE gel.
The following antibodies were used for immunoblotting: YKL-40 (Abcam, #ab77528), YKL-40 (Cell Signaling, #47066), Actin (Santa Cruz, #sc-1615), anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz, #SC-2352), anti-rabbit HRP-conjugated secondary antibody (Cell Signaling, #7074P2), and anti-mouse HRP-conjugated secondary antibody (Cell Signaling, #7076s).
The following antibodies were used for immunoblotting: YKL-40 (Abcam, #ab77528), YKL-40 (Cell Signaling, #47066), Actin (Santa Cruz, #sc-1615), anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz, #SC-2352), anti-rabbit HRP-conjugated secondary antibody (Cell Signaling, #7074P2), and anti-mouse HRP-conjugated secondary antibody (Cell Signaling, #7076s).
4
Purification and Evaluation of Isothiocyanates
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ITCs (SFN, 6-MSITC, and 6-MTITC) were purified from Wasabi by reversed-phase high performance liquid chromatography (HPLC) to >99.3% purity26 (link) and dissolved in dimethyl sulfoxide for cell culture experiments. The antibodies against Nrf2 (C-20), Keap1 (E-20), NQO1 (C-19), HSP70 (D69), GAPDH, rabbit IgG, and horseradish peroxidase (HRP)-conjugated anti-goat secondary antibody were purchased from Santa Cruz Biotechnology. AKR1C1, AKR1C3, and TXNRD1 antibodies were obtained from Abcam. HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology.
IMR-32 cell culture. Human neuroblastoma IMR-32 cells (cell no. TKG0207) were obtained from Riken Bioresource Center Cell Bank. IMR-32 cells were grown in Eagle’s Minimum Essential Medium (Nissui Seiyaku) supplemented with 2 mM l-glutamine (Nacalai Tesque), 1% v/v MEM nonessential amino acid solution (Nacalai Tesque), and 10% v/v fetal bovine serum (Equitech-Bio) under a humidified 5% CO2 atmosphere at 37 °C.
IMR-32 cell culture. Human neuroblastoma IMR-32 cells (cell no. TKG0207) were obtained from Riken Bioresource Center Cell Bank. IMR-32 cells were grown in Eagle’s Minimum Essential Medium (Nissui Seiyaku) supplemented with 2 mM l-glutamine (Nacalai Tesque), 1% v/v MEM nonessential amino acid solution (Nacalai Tesque), and 10% v/v fetal bovine serum (Equitech-Bio) under a humidified 5% CO2 atmosphere at 37 °C.
5
Detecting Kidney Injury Molecule-1 Expression
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