Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein extractions were harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After the incubation with a high affinity anti-METTL3 antibody (1:1000, Abcam, USA), anti-DGCR8 antibody (1:1000, Abcam, USA), anti-PTEN antibody (1:1000, Abcam, USA), anti-β-actin antibody (1:1000, Cell Signaling Technology, USA) or anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After washes, signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software.
Peroxidase hrp conjugated secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Peroxidase (HRP)-conjugated secondary antibody is a laboratory reagent that binds to primary antibodies, enabling the detection and visualization of target proteins in various immunoassays. The conjugated enzyme, horseradish peroxidase (HRP), catalyzes a colorimetric or chemiluminescent reaction, providing a signal that can be measured to quantify the presence and abundance of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Protease inhibitor, by Merck Group (4 mentions)
Anti gapdh antibody, by Cell Signaling Technology (3 mentions)
Chemiluminescence system, by Bio-Rad (3 mentions)
Bca analysis, by Beyotime (3 mentions)
Anti β actin antibody, by Cell Signaling Technology (2 mentions)
Anti pten antibody, by Abcam (1 mentions)
Anti mettl3 antibody, by Abcam (1 mentions)
Anti dgcr8 antibody, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemiluminescence system, by Merck Group (1 mentions)
Anti vimentin antibody, by Proteintech (1 mentions)
Control sirna, by Genechem (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
8 protocols using peroxidase hrp conjugated secondary antibody
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of TIPARP
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Tissues and cells treated with/without metformin were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After incubated with the anti-TIPARP antibody (1:1000, Abcam, USA) and anti-β-Actin antibody (1:1000, Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After washes, signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software.
3
Quantitative Protein Analysis by Western Blot
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Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein extracts were harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After incubation with a high-affinity anti-KRT6B antibody (1:2000, Proteintech, USA), anti-vimentin antibody (1:1000, Proteintech, USA) or anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After washing, the signals were detected using a chemiluminescence system (Millipore, USA) and analysed using Image Lab Software (Bio-Rad, USA).
4
Silencing MOXD1 in Fibroblasts
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SiRNA against human MOXD1 and control siRNA was purchased from GENE (Genechem, Shanghai, China). 2× 106 fibroblast cells were plated in 6 wells dishes and infected with 50nM of siRNA using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. 36h after transfection, cells were lysed in RIPA for Western blot. Total cellular proteins were lysed by RIPA buffer containing protease inhibitor. Protein concentration were estimated by the BCA (Thermo Scientific), 40μg were loaded per lane on 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocked with 5% fat-free milk, the membranes were incubated with anti-MOXD1 antibody (1:1000, ABCAm, USA) or anti-GAPDH antibody (1:1000, ABCAm, USA) at 4 °C overnight. The membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). Signals were visualized with Immobilon™ western chemiluminescent HRP substrate (Millipore) and analyzed by Image Lab Software.
5
Western Blot Analysis of RNA Modification Proteins
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Total protein was extracted using RIPA buffer (Beyotime, China) supplemented with a protease inhibitor cocktail (Yeason, China). The protein concentration was determined using the bicinchoninic acid (BCA) assay (Yeason, China). The protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were then incubated with primary antibodies against METTL3, METTL14, ALKBH5, FTO, MIB1 (all from Proteintech, Wuhan, China) at a dilution of 1:1000. The antibodies against HSV viral proteins ICP0, ICP8, and gC were obtained from Dr. Bernard Roizman’s laboratory at The University of Chicago and were used at a dilution of 1:1000. Subsequently, the membranes were incubated with a peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After thorough washing, the protein signals were detected using a chemiluminescence system (Tanon, China) and analyzed using Image Lab Software. The detailed information on the antibodies and related agents used in this study is provided in the Supplementary Table 2 .
6
Western Blot Quantification of RNA Methylation Regulators
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Western blotting experiments were performed in accordance with our previous study [29 (link)]. Briefly, tissues were harvested and lysed in RIPA buffer on ice, and the extracted protein was quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Next, proteins were resolved using 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Nonspecific binding sites were then blocked by immersing the membranes in 5% bovine serum albumin in PBS at room temperature. Membranes were then incubated with the following high affinity primary antibodies: anti-WTAP antibody (1:1000, Cell Signaling Technology, USA), anti-METTL3 antibody (1:1000, Cell Signaling Technology, USA), anti-METTL14 antibody (1:1000, Cell Signaling Technology, USA), and anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA). After washing, membranes were incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). Finally, protein signals were detected using a chemiluminescence imaging analysis system (Tanon, Shanghai, China).
7
Quantitative Protein Analysis Using Western Blot
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Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein concentration was quantified using a BCA Protein Assay kit (Pierce, USA). Total protein was separated using 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, USA). Western blot analysis followed a standard procedure. After incubated with the primary antibodies anti-WTAP, anti-CDK2 or anti-GAPDH (Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA). After washes, signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software.
8
Western Blot Analysis of Neuroinflammatory Markers
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