The exosome pellet was dissolved in radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4°C, and the protein concentration was determined using a Bradford protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (17 (link),18 (link)). The proteins (20 µg/lane) were separated via 15% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were blocked in 5% non-fat dried milk for 1 h, followed by incubation for 1 h with anti-CD9 (1:1,000; cat. no. 13174, Cell Signaling Technology, Inc.) and anti-heat shock protein 90α (HSP90α; 1:1,000; cat. no. 8165, Cell Signaling Technology, Inc.) primary antibodies, and subsequent incubation for 1 h with the secondary antibodies (horseradish peroxidase-conjugated anti-rabbit immunoglobulin G; 1:1,000; cat. no. 7074, Cell Signaling Technology, Inc.). The bands were visualized using the SuperSignal chemiluminescence system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All steps were performed at room temperature.
Horseradish peroxidase conjugated anti rabbit immunoglobulin g
Manufactured by Cell Signaling Technology
Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G is a secondary antibody used in various immunoassays and detection methods. It specifically binds to rabbit primary antibodies and is conjugated with the enzyme horseradish peroxidase, which can be used for signal amplification and visualization.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine peroxidase substrate kit, by Yeasen (2 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Supersignal chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Cell Signaling Technology (1 mentions)
Amersham hybond n membrane, by GE Healthcare (1 mentions)
Ribominus transcriptome isolation kit, by Thermo Fisher Scientific (1 mentions)
Miniprep kit, by Merck Group (1 mentions)
Hybond, by Cytiva (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Gerpn1210b, by Merck Group (1 mentions)
Rabbit anti m6a antibody, by Synaptic Systems (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
5 protocols using horseradish peroxidase conjugated anti rabbit immunoglobulin g
1
Western Blot Analysis of Exosomes
2
Quantifying mRNA m6A Modification
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mRNA was enriched using GenElute messenger RNA (mRNA) Miniprep Kit (Sigma‐Aldrich) and RiboMinus Transcriptome Isolation Kit (human/mouse; ThermoFisher Scientific). After 5 min of denaturation at 70 °C, equal amounts of serial-diluted mRNA were added into an Amersham Hybond N + membrane (GE Healthcare) and cross-linked into auto-cross-linker three times under auto-cross-linking mode. Membrane was blocked with 5% milk in 1× PBST for 1 h at room temperature and then was inducted with anti‐m6A antibody (SySy) overnight (4 °C). Membrane was washed according to the standard protocol. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Cell Signaling Technology) was diluted 1:5000 and incubated with the membranes for 1 h at room temperature. Membranes were developed with a 3,3′‐diaminobenzidine peroxidase substrate kit (Yeasen Biotechnology, Shanghai, China) to detect the signal.
3
Neonatal Cardiomyocyte Protein Extraction and Western Blot Analysis
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Total proteins were extracted from the neonatal cardiomyocytes or myocardium with the lysis buffer supplemented with 1mM PMSF, and a protease inhibitor cocktail. Protein in the supernatant was quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology, China). 20 to 50 μg of protein in each sample was subjected to polyvinlidene difluoride (PVDF) membranes. Blots were probed with specific antibodies against EGFR, phospho-EGFR (Y1068), ERK, phospho-ERK(1:1500), p38, phospho-p38(1:1000, Cell Signaling Technology, Danvers, MA, USA) respectively. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (1;2000; Cell Signaling Technology) was used as secondary antibody. The membranes were examined with a Kodak image station 2000R apparatus (Kodak, Rochester, NY, USA). β-actin was used as the control for equal loading of the protein.
4
Dot Blot Analysis of m6A RNA Modifications
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Dot blots were performed as follows. Total RNA was isolated. The RNAs (100 and 250 ng respectively) were double diluted and spotted onto a nylon membrane (Sigma-Aldrich, GERPN1210B). Then the membranes were ultraviolet (UV) crosslinked and blocked in blocking buffer (5% milk in phosphate-buffered saline with 0.1% Tween 20) for 1 h. Rabbit anti-m6A antibody (Synaptic Systems, 202003) was diluted 1:1,000 and incubated with the membranes overnight (4°C). After washing twice with 0.1% phosphate-buffered saline-Tween 20, horseradish peroxidase conjugated anti-rabbit immunoglobulin G (Cell Signaling Technology, 7074S) was diluted 1:5,000 and incubated with the membranes for 1 h at room temperature. After extensive washing, membranes were detected with a 3,3′-diaminobenzidine peroxidase substrate kit (Yeasen Biotechnology, 36302ES01). The same amount of poly(A)+ RNAs were spotted on the membrane, stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) for 2 h, and washed with ribonuclease-free water for 5 h.
5
Quantitative Western Blot Analysis
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We isolated total proteins from the cultured cells, and detected protein concentrations using a Pierce bicinchoninic acid protein assay kit (BCA Protein Assay Kit). We separated 20 μg protein from each sample using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred them electrophoretically to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk powder in phosphate-buffered saline (PBS) containing 0.1% Tween 20 at 37°C for 2 h, and then incubated overnight with primary antibodies against WT1 (1:1000; Cell Signaling Technologies, Beverly, MA, USA) and β-actin (1:5000; Cell Signaling Technologies). Horseradish peroxidase–conjugated anti-rabbit immunoglobulin G (Cell Signaling Technologies) was used as the secondary antibody. Bands were scanned using a densitometer (Bio-Rad, Richmond, CA, USA), and performed quantification using Bio-Rad Image Lab 4.1 software.
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