Western blotting was performed according to standard procedures using enhanced chemiluminescence detection. Briefly, total proteins were extracted from cells using RIPA lysis buffer (Thermo Fisher Scientific). Equal amounts of protein were separated on sodium dodecyl sulfate–polyacrylamide gels and then polyvinylidene fluoride membrane (Millipore). After blocking with 5% skim milk, the membranes were incubated with the primary antibodies for DNMT3A, DNMT3B, E-cadherin, N-cadherin, vimentin and GAPDH (Abcam), followed by incubation with corresponding horseradish peroxidase-coupled secondary antibody (Beyotime). The signals were detected with enhanced chemiluminescence reagents (Pierce). GAPDH was used as an internal control.
Horseradish peroxidase coupled secondary antibody
Manufactured by Beyotime
Sourced in China
Horseradish peroxidase-coupled secondary antibody is a laboratory reagent used in immunoassays. It consists of a secondary antibody conjugated with the enzyme horseradish peroxidase. The enzyme can catalyze a colorimetric reaction, allowing for the detection and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Dnmt3a, by Abcam (2 mentions)
Dnmt3b, by Abcam (2 mentions)
Vimentin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Bradford protein assay kit, by Beyotime (1 mentions)
Electrogenerated chemiluminescence reagent, by Merck Group (1 mentions)
Collagen 3, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab7778, by Abcam (1 mentions)
Enhanced chemiluminescence system, by Bio-Rad (1 mentions)
Tgf β, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ab64715, by Abcam (1 mentions)
Ab6992, by Abcam (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Sc 25357, by Santa Cruz Biotechnology (1 mentions)
7 protocols using horseradish peroxidase coupled secondary antibody
1
Western Blotting of EMT Markers
2
Protein Expression Analysis Protocol
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Conduction of Radio-Immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and Bradford Protein Assay Kit (Beyotime) was for protein extraction and concentration measurement. Isolation via 10% sulfate polyacrylamide gel electrophoresis of the protein sample (15 μg) and electroblot onto Polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) were implemented. Then incubation of the membrane was with 5% skim milk and test was with primary antibodies, consisting of ACTG1 (4968), E-cadherin (3195), N-cadherin (13,116), Snail (3879), GAPDH (2118) (all Cell Signaling Technology). Detection of the membrane was with horseradish peroxidase-coupled secondary antibody (Beyotime), followed by visualization with electrogenerated chemiluminescence reagent (Millipore). Application of GAPDH was as a loading control gene.
3
Liver Protein Expression Analysis
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Snap-frozen liver samples were homogenized in radioimmunoprecipitation assay buffer. Protein concentration was determined by BCA protein assay (Thermo Fisher Scientific). Equal amounts of protein were subjected to SDS-PAGE gels following with electrophoretic transfer to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk, and incubated with the primary antibody for Foxa3 (sc-25357; Santa Cruz), transforming growth factor-β (TGF-β, Ab64715; Abcam), CTGF (Ab6992; Abcam), Collagen I (Ab34710; Abcam), Collagen III (Ab7778; Abcam), α-smooth muscle actin (α-SMA, #14968; Cell Signaling Technology, Danvers, MA, USA) or GAPDH (#5174, Cell Signaling Technology). After incubated with corresponding horseradish peroxidase-coupled secondary antibody (Beyotime, Shanghai, China), the membrane was developed with enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA). Densitometric analysis was performed by using ImageJ software (National Institutes of Health, Bethesda, MD, USA) using GAPDH as an endogenous control.
4
Protein Expression Analysis in Liver
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Snap-frozen liver samples and cells were homogenized in RIPA lysis buffer (Thermo Fisher Scientific, Hudson, NH, USA). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed with wet transfer to nitrocellulose membranes. The membranes were blocked with 5% skim milk, and incubated with the primary antibodies for the DNMT1, DNMT3a, DNMT3b, IFN-γ and β-actin proteins (Abcam, Cambridge, MA, USA). After being incubated with a corresponding horseradish peroxidase-coupled secondary antibody (Beyotime, Shanghai, China), the membrane was allowed to react with the Lumi-Light ECL substrate (Thermo Fisher Scientific). ImageJ was then used to analyze the western blot result.
5
Western Blot Analysis of Protein Expression
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The cells were lysed in lysis buffer (Promega Corporation) and centrifuged at 4˚C, 12,000 x g for 10 min. The supernatant was collected and subjected to western blotting. Protein concentration was subsequently measured using a BCA Protein Assay kit (cat. no. P0012; Beyotime Institute of Biotechnology). Protein (50 µg) from each lysate was fractionated by 10% SDS-PAGE. The samples were electrophoresed for 2 h and transferred onto polyvinylidene difluoride membranes (MilliporeSigma). After being blocked with 5% BSA in TBS-0.1% Tween-20 (TBST) for 1 h at room temperature, the membranes were blotted with HuR (Abcam; cat. no. ab220342) or α-Tubulin (Beyotime Institute of Biotechnology; cat. no. AF0001) primary antibodies at 1:1,000 dilutions. The membranes were then incubated with the appropriate horseradish peroxidase-coupled secondary antibody (Beyotime Institute of Biotechnology; cat. no. A0277) at a 1:2,000 dilution for 1 h at room temperature. After the membranes were washed with TBST, the blots were incubated with enhanced chemiluminescence (ECL) stable peroxide solution (Beyotime Institute of Biotechnology). All blots were visualized using a FluoroChem MI imaging system (Alpha Innotech Corporation) at room temperature. Densitometry was performed using ImageJ v1.8.0-172 software (National Institutes of Health).
6
CaMKII Phosphorylation in Hippocampus
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Levels of total CaMKIIα and pCaMKIIα (CaMKII phosphorylated at T286) were investigated in hippocampi of experimental rats (n = 4 for each group). Each hippocampus was homogenized in lysis buffer (pH 7.4, 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin, etc.) and centrifuged at 12,000g for 15 min. After being diluted and denatured, the samples (about 20 μg) were separated by SDS-PAGE (10% resolving gel), transferred to a PVDF membrane and blocked for 1 h. Then the membrane was incubated with mouse polyclonal anti-CaMKII antibody (1:400, Santa Cruz Biotechnology) or rabbit polyclonal anti-pCaMKII antibody (1:400, Santa Cruz Biotechnology) overnight at 4 °C. After incubation with a horseradish peroxidase-coupled secondary antibody (1:3000, Beyotime, China) for 2 h at room temperature, the bands were visualized using the ECL detection system. The value of the band was calculated using the Bio-Rad video imaging system and expressed as a percentage of GAPDH.
7
NLRP3 Protein Expression Analysis
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HMC3 cells were lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) to extract the total protein. The protein samples were mixed with loading buffer and then denatured in the boiling water for 10 min. Next, the total protein was separated by SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, MA, USA). After that, the membranes were blocked in 5% defatted milk, and then incubated with the primary antibodies: anti-NLRP3 (1:500, ab214185, Abcam, Shanghai, China) and anti-β-actin (1:1000, ab8226, Abcam, Shanghai, China), respectively, overnight at 4°C. After the membranes were rinsed by TBST, the membrane was incubated with horseradish peroxidase coupled secondary antibody (Beyotime, Shanghai, China) at room temperature for 1 h. Finally, the protein bands were developed with ECL kit (Beyotime, Shanghai, China).
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