Protein was extracted using RIPA-reagent LS (Aidlab Biotechnologies, Beijing, China) according to the manufacturer’s protocols. Protein level was measured using a Bicinchoninic Acid kit (Thermo Scientific). Then, 40 μg of protein lysate was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and then immunoblotted with antibodies against phospho-AMPKα1 (Thr-172; 1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), AMPK (1:1000; Abcam, Cambridge, UK), PPARα (1:1000; Abcam), and GAPDH (1:2000; Abcam). Subsequently, they were incubated with horseradish peroxidase-conjugated secondary antibodies (MultiSciences (Lianke) Biotech, Hangzhou, China), visualized using an ECL Plus kit (Millipore) and exposed by autoradiography (Eastman Kodak, Rochester, NY, USA).
Horseradish peroxidase conjugated secondary antibody
Manufactured by MultiSciences Biotech
Sourced in China
Horseradish peroxidase-conjugated secondary antibodies are laboratory reagents used in various immunoassays and detection techniques. They consist of secondary antibodies that are chemically linked to the enzyme horseradish peroxidase. This enzyme-antibody conjugate can be used to detect and visualize the presence of target proteins or other biomolecules in samples.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Enhanced chemi luminescence (ecl), by Sartorius (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ecl plus kit, by Merck Group (1 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Pparα, by Abcam (1 mentions)
Ripk3, by Abcam (1 mentions)
P ripk1 ser166, by Cell Signaling Technology (1 mentions)
P mlkl ser358, by Abcam (1 mentions)
Ecl detection kit, by Sartorius (1 mentions)
Bcr abl, by Cell Signaling Technology (1 mentions)
Ripk1, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti histone h1, by Cell Signaling Technology (1 mentions)
Anti phospho gsk3β, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
8 protocols using horseradish peroxidase conjugated secondary antibody
1
Western Blot Analysis of AMPK and PPARα
2
Western Blot Analysis of Protein Signaling
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Cultured cells were lysed at 4 °C in lysis buffer and protein concentrations determined by the bicinchoninic acid (BCA) method. Equal amounts of total cell lysates were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels (8-12% gradient) and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked for 2 h with Tris-buffered saline containing 0.1% Tween and 5% nonfat dry milk and incubated with primary antibodies overnight at 4 °C. After incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Multi Sciences Biotech), immunoreactions were visualized with an ECL detection kit (Biological Industries, Beit HaEmek, Israel). Primary antibodies specific for the following proteins were used: RIPK1, p-RIPK1 (Ser166), BCR/ABL, p-BCR/ABL (Tyr177), and β-actin (Cell Signaling Technology, CST; Beverly, MA, USA); RIPK3, p-RIPK3 (Ser223), MLKL, and p-MLKL (Ser358) (Abcam, Cambridge, UK).
3
Protein extraction and Western blotting
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Total protein was extracted from the collected cells. Cytoplasmic and nuclear proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Equal amounts of protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were then transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). Membranes were subsequently blocked for 2 h in Tris-buffered saline containing 0.1% Tween and 5% nonfat dry milk followed by incubation with primary antibodies overnight at 4°C. After a second incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Multi Sciences Biotech, Hangzhou, China), the bands on the PVDF membranes were developed using enhanced chemiluminescence (ECL; Biological Industries, Beit HaEmek, Israel). All primary antibodies (anti-γ-catenin, anti-β-catenin, anti-Cyclin D1, anti-c-Myc, anti-GSK3β, anti-phospho-GSK3β, anti-Histone H1 and anti-β-actin) were obtained from Cell Signaling Technology (Beverly, MA, USA).
4
Western Blot Protein Expression Analysis
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Western blotting was performed using a standard protocol. Briefly, HCC cells were harvested and lysed with 1% ice-cold SDS lysis buffer (Beyotime, Shanghai, China) include protease and phosphatase inhibitors (Roche), and protein concentration was quantified using a BCA Assay Kit (Thermo Fisher, DE, USA). Protein samples (30 μg) were subjected to 4-20% gradient PAGE gel (GenScript M42012) with Tris-MOPS running buffer, and transferred onto PVDF membranes (Millipore, MA, USA). The membranes were blocked with 5% non–fat milk at RT for 1 h and incubated sequentially with primary antibodies at 4° C overnight. Horseradish peroxidase-conjugated secondary antibodies (MultiSciences, Hangzhou, China) were added the following day for 1 h and then washed with TBST. Protein signals were detected using chemiluminescent ECL reagent (Thermo Scientific, USA), analyzed on an AlphaImager HP gel imaging system, and quantified with an image analysis software.
5
Western Blot Analysis of Apoptosis and Autophagy
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Total proteins were extracted from the collected cells. Equal amounts of protein were separated via SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). The membranes were subsequently blocked for 2 h in Tris-buffered saline containing 0.1% Tween and 5% nonfat dry milk and then incubated with primary antibodies overnight at 4 °C. After incubation with horseradish peroxidase-conjugated secondary antibodies (1:5000; Multi Sciences Biotech, Hangzhou, China), the PVDF membranes were visualized using chemiluminescence (ECL; Biological Industries, Beit Ahemeq, Israel). The specific Caspase-3 inhibitor Z-DEVD-FMK (DEVD) and the tyrosine kinase inhibitor imatinib were obtained from Selleck Chemicals. The primary antibodies used were as follows: anti-BECLin1, anti-cleaved PARP, anti-cleaved Caspase-3, anti-Atg5, anti-Atg7, anti-UVRAG, anti-p-BCR/ABL (Tyr177), anti-Flag, anti-HA, and anti-GAPDH, purchased from Cell Signaling Technology (Beverly, MA, USA), and anti-BCR/ABL, anti-p62/SQSTM1, and anti-LC3 I/II, purchased from Abcam (Cambridge, UK).
6
Protein Extraction and Western Blot Analysis
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RIPA Lysis Buffer (Applygen Technologies Inc., Beijing, China) was used to extract the total proteins from the SHSY5Y cells. A BCA kit (MultiSciences, Hangzhou, China) was used to measure the protein concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (15% or 8%, MultiSciences) was used for target protein separation, and the polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany) were then transferred and blocked with 5% skimmed milk powder at 25 ℃ for 90 min. The membranes were incubated overnight at 4 ℃ with the following primary antibodies: anti-Bad (ab32445, 1:1,000), anti-Bcl-2 (ab182858, 1:2,000), anti-Cleaved caspase-3 (ab2302, 1:400), anti-extracellular regulated protein kinase (ERK) (ab184699, 1:8,000), anti-phospho-ERK (ab201015, 1:1,000) (Abcam, Cambridge, UK), anti-protein 38 (p38) (#9212, 1:1,000, CST), anti-phospho-p38 (#9216, 1:2,000, CST), and glyceraldehyde-3-phosphate dehydrogenase (ab8245, 1:8,000, Abcam). The membranes were then incubated with horse radish peroxidase-conjugated secondary antibodies (1:1,000, MultiSciences) at 25 ℃ for 90 min. Next, the bands were visualized.
7
Proteomic Analysis of Extracellular Vesicles
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Western blotting was performed as described previously [25 (link)]. EXOs and cell lysates were immunoblotted with these antibodies: anti-CD63, CD81, TSG101, Alix, Cytochrome C, Hsp70, MMP-2, MMP-9, CTLA-4, and c-Myc (Abcam, Cambridge, MA); anti-Bcl-2, Bcl-xl, Mcl-1, xIAP, TGF-β, TRAIL, GSK-3β, and FASL (Cell signaling technology, Beverly, MA); anti-FAS and BTLA (Proteintech, Rocky Hill, NJ, USA). The horseradish peroxidase-conjugated secondary antibody was obtained from MultiSciences Biotech, Hangzhou, China. Immunoblotting with anti-actin or anti GAPDH (Cell signaling technology) confirmed equivalent protein loading.
8
Renal Tissue Protein Extraction and Analysis
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The proteins in the renal tissues were extracted with lysis buffer. After denaturation, 40 μg of protein was loaded on the 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred to polyvinylidene fluoride membranes. After blocking overnight at 4 °C with 4% skimmed milk, the membranes were incubated with the anti-GAPDH (Multi Sciences, Hangzhou, China) and anti-PTEN (Multi Sciences, Hangzhou, China) antibodies. After washing, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (Multi Sciences, Hangzhou, China) to identify the respective proteins. The bands were visualized using enhanced chemiluminescence (Meilun, Shanghai, China) and quantified with Image J software. GAPDH was used as an internal control.
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