Total proteins were extracted using Radio-Immunoprecipitation Assay (RIPA) Lysis Buffer (Beyotime, Shanghai, China) according to the manufacturer's instructions. The concentration of total proteins was determined using the BCA Protein Assay Kit (Bio-Rad; Hercules, CA, USA). Equal amounts of protein (approximately 30μg per sample) were denatured prior to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoreses, then electro-transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocked with 5% skimmed-milk in TBST for 60 min, the membranes were incubated overnight at 4°C with rabbit anti-SMOC2 (1:200 dilution; Abcam, Cambridge, MA, USA) and rabbit anti-GAPDH (1:8000 dilution; Proteintech, Chicago, IL, USA). Then, the membranes were washed four times for 10 min with TBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:2000 dilution; Cell Signaling Technology, Danvers, MA, USA) for 1h at room temperature. The membranes were washed four times with TBST and the immunoreactive proteins were visualized using an enhanced chemiluminescence system (Cell Signaling Technology). The band intensities were measured using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) and target protein levels in each sample were expressed relative to GAPDH.
Horseradish peroxidase hrp conjugated goat anti rabbit antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody is a secondary antibody used in various immunoassays and detection techniques. It consists of a goat-derived antibody that binds to rabbit primary antibodies, with a covalently attached horseradish peroxidase enzyme. The HRP enzyme can be used to catalyze a colorimetric or chemiluminescent reaction, enabling the detection and visualization of target proteins or molecules in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence system, by Cell Signaling Technology (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Beyotime (1 mentions)
Rabbit anti gapdh, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Occludin, by Santa Cruz Biotechnology (1 mentions)
Zonula occludens 1 zo 1, by Santa Cruz Biotechnology (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
β actin, by Sungene Biotech (1 mentions)
Cleaved caspase 3, by Merck Group (1 mentions)
α tubulin, by Merck Group (1 mentions)
Epiquik in situ dna damage assay kit, by Epigentek (1 mentions)
Nf κb, by Merck Group (1 mentions)
Dna damage quantification colorimetric kit, by Abcam (1 mentions)
Hrp conjugated goat anti mouse antibody, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti mouse antibody, by Cell Signaling Technology (1 mentions)
Cleaved caspase 7, by Cell Signaling Technology (1 mentions)
9 protocols using horseradish peroxidase hrp conjugated goat anti rabbit antibody
1
Quantification of SMOC2 Protein Levels
2
Molecular Mechanisms of Intestinal Barrier Regulation
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Sal was obtained from TCI Chemical Industry Co., Ltd. (Shanghai, China). DSS (molecular weight of 36–50 kDa) was purchased from MP Biomedicals (Irvine, CA, USA). The primary antibodies p38, p-p38, p65, p-p65, and the secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The secondary antibody HRP-conjugated goat anti-rabbit and goat anti-mouse antibody were obtained from Immunoway (Immunoway Technology, USA). The primary antibodies occludin and zonula occludens-1 (ZO-1) were purchased from Santa Cruz (Santa Cruz, CA, USA). β-Actin were purchased from Tianjin Sungene Biotech Co., Ltd. (Tianjin, China). All enzyme-linked immunosorbent assay (ELISA) kits were obtained from BioLegend (San Diego, CA, USA). Protein Extraction Kit was provided by Thermo Scientific Life Science Research (MA, USA). All other chemicals were of reagent grade.
3
Apoptosis and DNA Damage Assessment
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The FITC-labelled Annexin V Apoptosis Detection Kit was obtained from BD Biosciences (San Jose, CA, USA). DNA Damage Quantification Colorimetric Kit was purchased from Biovision (Milpitas, CA, USA) and EpiQuik In Situ DNA Damage Assay kit was obtained from Epigentek (Farmingdale, NY, USA). JC-1, cisplatin, and DCFH-DA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The culture medium was obtained from Invitrogen Technologies (Carlsbad, CA, USA). The antibodies were obtained from the following sources: TRAF6 and MVP from Abcam (Cambridge, UK); α-tubulin from Sigma-Aldrich; p62, NFκB, cleaved-PARP, cleaved-caspase 3, cleaved-caspase 7, cleaved-caspase 9, IKKβ, IKKα, phosphor-IKKβ, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody, HRP-conjugated goat anti-mouse antibody, and Alexa Fluor 488-conjugated goat anti-mouse antibody from Cell Signalling Technology (Danvers, MA, USA).
4
Molecular Mechanisms Regulating Ca2+ Homeostasis
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HMGB1, PP2, CGP77675, SKF96365, 2-APB, thapsigargin (TG) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fluo-4/AM and CCK-8 Kits were purchased from Dojindo Laboratories (Kumamoto, Japan). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from Gibco (Grand Island, NY, USA). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA). FITC-labeled secondary antibodies, mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody and 4',6-diamidino-2-phenylindole (DAPI) were from Santa Cruz Biotechnology (CA, USA). Rabbit polyclonal anti-VE-cadherin antibody, rabbit polyclonal anti-Na,K-ATPase α1 antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody, rabbit polyclonal anti-phospho-Src antibody and rabbit polyclonal anti-Src antibody were from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal anti-STIM1 antibody, and anti-human Orai1 antibody were from Abcam (Cambridge, MA, USA).
5
Investigating Nicotinic Receptor Signaling
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The FITC-labeled Annexin V Apoptosis Detection Kit was obtained from BD Biosciences (Canada). The ROS assay kit (DCFH-DA) was purchased from Meilun (China). The cell culture medium was obtained from HyClone (Utah, USA), and cell-cultured grade fetal bovine serum (FBS), penicillin/streptomycin, and trypsin were purchased from Gibco (Thornton, Australia). The antibodies of p-Erk1/2, Erk1/2, p-Akt, Akt, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9, β-actin, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-nicotinic acetylcholine receptor α7 antibody was purchased from Abcam (ab216485, Abcam). The drugs were obtained from the following sources: nicotine, methyllycaconitine citrate (MLA), dihydro-β-erythroidine hydrobromide (DHβE), and PD0325901 from MedChemExpress (MCE, USA) and H2O2 and N-acetylcysteine (NAC) from Sigma–Aldrich (St. Louis, MO, USA).
6
Western Blot Analysis of Skeletal Muscle
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The skeletal muscle tissue (EDL) was homogenized using RIPA lysis buffer (Thermo, 89,900, IL, USA) supplemented with Halt™ Protease and Phosphatase Inhibitor Single-Use Cocktail (Thermo, 1,861,280, IL, USA). The resulting supernatant was collected, and the total protein content was measured using the Rapid Gold BCA Protein Assay Kit (Thermo Scientific, A53226, IL, USA) according to the manufacturer’s instructions. Proteins (25 µg) were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels ranging from 8 to 12%. The separated proteins were then transferred onto polyvinylidene difluoride membranes (Bio-Rad, 1,620,177, CA, USA) and blocked with 5% skimmed milk (Bio-Rad, 1,706,404, CA, USA) for 2 h. Afterward, the membranes were washed three times with TBST and incubated overnight at 4 °C with primary antibodies. The primary antibodies used in this study included AKT, p-AKT (Ser473), mTOR, p70S6K, p-p70S6K, and p-FOXO3A (all from Cell Signaling Technology, MA, USA; 1:1000), as well as FOXO3A, FOXO1, NF-κB, and MuRF1 (all from Proteintech, Wuhan, China; 1:1000). A secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA; 1:2000), was applied. The protein bands were visualized using the ChemiDoc system (Bio-Rad, California, USA) and quantified using Image J software.
7
Analysis of A1M and HO-1 Protein Expression
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To analyze A1M secretion into the cell culture medium, equal volumes of cell culture supernatants were electrophoresed on 12% Tris-glycine SDS-PAGE gels, then proteins were transferred to 0.22 µm nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) and blocked with 5% w/v milk for 60 min; they were then probed for A1M antibody (Abcam, Cambridge, UK; dilution 1:2500) at 4 °C overnight, followed by incubation with horse-radish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technologies, Danvers, MA, USA) at room temperature for 1 h. The antigen–antibody complex was detected by a WesternBright ECL HRP substrate (Advansta, Menlo Park, CA, USA). To ascertain equivalent protein loading in the samples, membranes were stripped and reprobed again with a mouse anti-bovine serum albumin antibody (Abcam, Cambridge, UK). To analyze HO-1 expression, cells were lysed with a radioimmunoprecipitation assay buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 1% Sodium-deoxycholate, 0.1% SDS, 1 × Complete Mini Protease Inhibitor Cocktail) and analyzed by immunblot, as described above, using HO-1 antibody (Proteintech Group, Manchester M3 3WF, UK; dilution 1:8000), then reprobed with a mouse GAPDH antibody (Proteintech Group, Manchester, UK; dilution 1:10,000).
8
Investigating miRNA-34c-3p Regulation of PLCXD3
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The cells (GC-1, TM4 and NCM460 cells) were transfected with the indicated miRNA mimics and inhibitors (hsa-miR-34c-3p mimic: 5′-aaucacuaaccacacggccagg-3′, hsa-miR-34c-3p mimic inhibitor: 5′-ccuggccgugugguuagugauu-3′; negative control mimic: 5′-uuuguacuacacaaaaguacug-3′; negative control inhibitor: 5′-caguacuuuuguguaguacaaa-3′). Forty-eight hours later, the cells were lysed in RIPA buffer (Norgen-Biotek Corp.) containing 1× Halt Protease Inhibitor Cocktail (Pierce Inc., Rockford, IL, USA). Forty micrograms of total protein were separated and blotted using the Bio-Rad V3 Western work flow system, according to the manufacturer's recommendation. Immunoblotting was conducted by incubating the membrane with an anti-PLCXD3 rabbit polyclonal antibody (1:500, Santa Cruz Biotechnology, sc-137672) overnight at 4°C. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (1:3,000, Cell Signaling, 7074) was used as the secondary antibody, whereas an HRP-conjugated anti-β-tubulin antibody (1:10,000, Abcam, ab6046) was used as the loading control.
9
MTT Assay with Antibody Detection
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3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was purchased from Dojindo (Kumamoto, Japan). Rabbit anti-TLR4 antibody was acquired from Cell Signalling Technology (cat # 14358S; Danvers, MA, USA), and mouse anti-β-actin antibody was purchased from Santa Cruz Biotechnology (cat # sc-69879; Dallas, TX, USA). Alexa 488-conjugated goat anti-mouse IgG was procured from Cell Signalling Technology (cat # 4408S). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody and HRP-conjugated horse anti-mouse antibody were purchased from Cell Signalling Technology (cat # 7074S, 7076S).
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