SGC7901 cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration in cell lysates was measured using bicinchoninic acid (BCA) kit (KeyGen Biotechnology, Nanjing, Jiangsu, China). Then equal amounts of protein samples were mixed with loading buffer and boiled for 10 min for denaturation. The samples were subjected to 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking in 5% skim milk for 1 h, the 1 : 1000 diluted specific primary antibodies against FBXL2 and GAPDH (Abcam, Cambridge, MA, USA) were added onto membranes, followed by incubation at 4 °C overnight. On the next day, the 1 : 5000 diluted horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (Abcam) was added onto the membranes and incubated for 1 h at 37 °C. The enhanced chemiluminescence (ECL) reagent was used to develop the bands. The optical density of targeted bands was determined using Image J software (NIH, Bethesda, MD, USA). The experiment was performed in triplicate.
Horseradish peroxidase hrp labeled goat anti rabbit secondary antibody
Manufactured by Abcam
Sourced in United States
Horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody is a detection reagent used in various immunoassays and immunohistochemical techniques. It binds to and labels rabbit primary antibodies, enabling the visualization and detection of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca kit, by Keygen Biotech (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Azure c600 scanner, by Azure Biosystems (1 mentions)
Ibind flex western device, by Thermo Fisher Scientific (1 mentions)
Ibind flex solutions, by Thermo Fisher Scientific (1 mentions)
Criterion precast, by Bio-Rad (1 mentions)
Naphthol as d chloroacetate specific esterase kit, by Merck Group (1 mentions)
Lenalidomide, by MedChemExpress (1 mentions)
Tnf α, by MedChemExpress (1 mentions)
Recombinant tnf α, by Thermo Fisher Scientific (1 mentions)
Sp600125, by MedChemExpress (1 mentions)
Abt 199, by MedChemExpress (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Elisa, by BD (1 mentions)
Diaminobenzidine dab kit, by ZSGB-BIO (1 mentions)
Fibronectin, by Abcam (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Alpha smooth muscle actin α sma, by Abcam (1 mentions)
6 protocols using horseradish peroxidase hrp labeled goat anti rabbit secondary antibody
1
Western Blot Analysis of FBXL2 Expression
2
Apoptosis-related Protein Expression Assay
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For expression level determination of apoptosis-related proteins Caspase-3, Bcl-2, Bax, cells were treated with GEM and M1-Exo-GEM for 24 h, lysed on ice with 1.0 ml of RIPA lysate (Beyotime Biotechnology, Shanghai, China) for 30 min, and then centrifuged at 10,000 g for 15 min, followed by collection of supernatant for protein quantification. Next, SDS-PAGE gel electrophoresis was performed at 80V–120 V, and membranes were transferred at a constant 300 mA for 0.5–2 h. The PVDF membranes were blocked with 5% skimmed milk powder for 2 h, and incubated overnight at 4° C with Caspase-3, Bcl-2 and Bax monoclonal primary antibodies (1:500 dilutions with TBST), and then with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (1:2000 dilution; Abcam, USA). Afterwards, chemiluminescent immunoassay was performed, and the optical density (OD) was analyzed via Image Pro-Plus 6.0 software. The results were expressed as OD comparisons between the target proteins and the internal reference (GAPDH).
3
Vitamin D Receptor Protein Analysis
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Protein was extracted from pancreatic cancer cells following cell washing and lysis. Next, 20 μg of each sample was fractionated on a Criterion Precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was stained with anti-VitD receptor antibody (Cell Signaling Technologies, Danvers, MA) and horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (Abcam, Cambridge, MA, USA) diluted 1:1,000 and 1:5,000, respectively, in iBind Flex solutions (Invitrogen, Waltham, MA, USA) overnight using the iBind Flex Western device (Thermo Fisher, Waltham, MA, USA) per the manufacturer’s protocol. The membrane was then scanned using the Azure C600 scanner (Azure Biosystems, Dublin, CA, USA).
4
Investigating TNF-α-Induced Inflammatory Response
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The materials are as follows: recombinant TNF-α (PeproTech, NJ, USA); ABT-199, SP600125, and lenalidomide (MedChemExpress, NJ, USA); TNF-α, myeloperoxidase (MPO), interleukin-1 β (IL-1β), IL-6, IL-8, and IL-18 enzyme linked immunosorbent assay (ELISA) kits (BD Biosciences, CA, USA); rabbit anti-mouse FoxO3a antibody, rabbit anti-mouse Ly6G antibody, rabbit anti-mouse JNK antibody, rabbit anti-mouse Bcl-2 antibody, rabbit anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, horseradish peroxidase- (HRP-) labeled goat anti-rabbit secondary antibody (Abcam, Cambridge, UK); HRP-labeled anti-mouse secondary antibody, bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Biotechnology, Shanghai, China); Naphthol AS-D Chloroacetate (Specific Esterase) Kit (Sigma, NY, USA); Diaminobenzidine DAB Kit (ZLI9019; ZSGB-BIO); Dulbecco's modified Eagle's medium (DMEM) medium, trypsin, and fetal bovine serum (FBS) (Gibco, NY, USA); FoxO3a small interfering ribonucleic acid (siRNA) (Guangzhou RiboBio Co., Ltd., Guangzhou, China); and Cell counting kit-8 (CCK-8) and Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) Apoptosis Assay Kit (Jiangsu KeyGEN BioTECH Corp., Ltd., Jiangsu, China).
5
Quantitative Protein Analysis of HCF Cells
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Total proteins extracted from sample HCF cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio) were quantified with bicinchoninic acid (BCA) protein assay kits (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. After the separation with 8% SDS-PAGE, the membranes were transferred onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% non-fat milk and then incubated with primary antibodies specific to connective tissue growth factor (CTGF; cat. no. ab209780; 1:1000; Abcam), Fibronectin (cat. no. ab268020; 1:1000; Abcam), alpha-smooth muscle actin (α-SMA; cat. no. 14395-1-AP; 1:1000; Proteintech), KLF5 (cat. no. ab137676; 1:1000; Abcam), phosphorylated (p)-p38 (cat. no. ab178867; 1:1000; Abcam), p-c-Jun N-terminal kinase (p-JNK; cat. no. ab307802; 1:1000; Abcam), p38 (cat. no. ab170099; 1:1000; Abcam), JNK (cat. no. ab199380; 1:2500; Abcam), or GAPDH (cat. no. ab9485; 1:2500; Abcam) at 4 °C overnight, following which was the incubation with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (cat. no. ab6759; 1:5000; Abcam) at room temperature for 2 h. Finally, the protein bands were visualized with enhanced chemiluminescence (ECL) Detection Reagent (Yeasen Biotech) and analyzed with Image J software (Version 1.8.0).
6
Western Blot Analysis of Apoptosis Regulators
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The abovementioned cell protein liquid extracted after culture was used for Western blotting (WB) assay. In brief, the 8%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel was prepared; then the protein liquid was diluted with 5× loading buffer to a volume of 20 μl. After being boiled for 8 min, the sample was loaded for electrophoresis at the voltage of 80 and then 120 V and transferred onto the membrane at the constant current of 300 mA for 0.5–2 h. Later, the membrane was blocked with 5% skimmed milk powder for 2 h and incubated with TBST-diluted monoclonal primary antibodies (1:400; Abcam, Massachusetts, USA) against Bcl-2, TGF-β1, NLRP3, ASC, and TNFR1. Afterward, the membrane was further incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (Abcam, USA). Finally, the membrane was detected with chemiluminescence, and the OD value was analyzed using the Image Pro-Plus 6.0 software.
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