The protein expressions of Bax, Bcl-2, and Bcl-xL were analyzed by western blotting. HepG2 cells were collected by centrifugation and resuspended in extraction buffer of mammalian protein extraction kit (Sangon, China). Cell debris was removed by centrifugation, and the supernatant was collected to detect the protein concentration by BCA method (Pierce, USA). An amount of 40 mg cellular protein was electro-blotted onto a PVDF membrane following separation on a 10 % SDS-polyacrylamide gel electrophoresis. The PVDF membrane was transferred to incubate with blocking solution (5% skim milk) for 2 hours at room temperature, and then incubated overnight with a 1:1000 dilution of anti-Bax, anti-Bcl-2, anti-Bcl-xL, anti-β-actin antibody at 4 °C. Blots were washed five times with Tween 20/Tris-buffered saline (TTBS) and then incubated with a 1:1000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Blots were washed five times with TTBS again and then developed by Horseradish peroxidase substrate (Millipore Corporation, USA) and data were captured by exposure to Kodak BioMax Light films.
Horseradish peroxidase substrate
Manufactured by Merck Group
Sourced in United States
Horseradish peroxidase (HRP) is an enzyme that catalyzes the oxidation of various substrates in the presence of hydrogen peroxide. HRP substrates are chemical compounds that can be used to detect and quantify the presence of HRP in various applications, such as immunoassays and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (3 mentions)
Biomax light film, by Kodak (2 mentions)
Imagequant las 4000 min ccd camera, by GE Healthcare (2 mentions)
Las 4000, by Cytiva (2 mentions)
Proteome profiler array kit, by R&D Systems (2 mentions)
Mammalian protein extraction kit, by Sangon (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Ab179515, by Abcam (1 mentions)
Ab180799, by Abcam (1 mentions)
Ab215203, by Abcam (1 mentions)
Ab93015, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Minigel, by Bio-Rad (1 mentions)
Gapdh, by Wuhan Servicebio Technology (1 mentions)
Goat anti mouse secondary antibody, by Wuhan Servicebio Technology (1 mentions)
Mini trans blot apparatus, by Bio-Rad (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Phospho iκbα, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
18 protocols using horseradish peroxidase substrate
1
Bax, Bcl-2, and Bcl-xL Protein Expression
2
Protein Extraction and Western Blot Analysis
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The cells were washed three times with cold PBS and collected by centrifugation and washed once with PBS. The washed cell pellets were resuspended in extraction lysis buffer (50 mM HEPES pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 1 mM PMSF, 0.5 mM DTT, 5 mM NaF and 0.5 mM sodium orthovanadate) containing aprotinin and leupeptin (5 mg/mL), and incubated for 20 min at 4°C. Cell debris was removed by centrifugation, and supernatants were rapidly frozen. The protein was detected by BCA method (Pierce, USA). Of cellular protein, 40 mg protein from cell extracts was electro-blotted onto a polyvinylidene difluoride (PVDF) membrane following separation on a 10% SDS-polyacrylamide gel electrophoresis. The immunoblot was incubated for 1 h with blocking solution (5% skim milk) at room temperature, and then incubated overnight with a 1:500 dilution of anti-BMP-2, anti-OCN, anti-Col I, β-actin antibody (Cell Singaling Technology, USA) at 4°C. Blots were washed three times with Tween 20/Tris-buffered saline (TTBS) and then incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA) for 1 h at room temperature. Blots were washed five times with TTBS and then developed by Horseradish peroxidase substrate (Millipore Corporation, USA) and data were captured by exposure to Kodak BioMax Light films.
3
Western Blot Analysis of Apoptosis-related Proteins
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The specific steps of the western blot assay were performed, as previously described [22 (link), 33 (link)] with the following antibodies: anti-Caspase-1 (ab179515), anti-GSDMD (ab219800), anti-GSDMD-N (ab215203), anti-ASC (ab180799) and anti-AIM2 (ab93015) (Abcam, Cambridge, UK); anti-γH2AX (9718) (CST, Danvers, MA, USA); anti-PIF1 (19006-1-AP), anti-BRCA1 (22362-1-AP), anti-GAPDH (60004-1-Ig), and the secondary antibodies (Proteintech, Wuhan, China). Blots were conjugated with the chemiluminescent horseradish peroxidase substrate (Millipore), visualized, and quantified by Quantity 5.2 (Bio-Rad) according to the manufacturers’ instructions.
4
Western Blot Analysis of Vasa in Ovarian Tissue
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Ovaries (50) were homogenized in 100 μl of 1× radioimmunoprecipitation assay buffer to prepare crude lysate. Protein lysate was separated on 12% denaturing sodium dodecyl sulfate polyacrylamide gel and transferred onto ImmunoBlot polyvinylidene difluoride membranes (Bio-Rad). Blots were washed with TBST (50mM Tris Base, 150mM NaCl, 0.1% Tween-20), followed by incubation in the blocking buffer (4% skimmed milk in TBST) for 30 min. Blots were probed with mouse anti-Vasa antibody (1:2000) (DSHB). After washing three times in TBST and another blocking for 30 min in the blocking solution, rabbit anti-rat IgG peroxidase conjugated (1:1500) (Bangalore GeNei) was added for 90 min, followed by three washes in TBST. Blots were exposed to a chemiluminescent horseradish peroxidase substrate (Millipore) in a dark room, and signals were documented on X-ray film.
5
Synaptotagmin-1 Expression Analysis
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The frozen spinal cord L4–6 was ground and mixed with cold RIPA Buffer (200 µL per 20 mg of tissue) (Beyotime Biotech, SH, China). Forty micrograms of proteins were subjected to 10% SDS-PAGE and transferred to a PVDF membrane using a minigel and mini transblot apparatus (Bio-Rad, Hercules, CA, USA). After being incubated in 5% nonfat milk containing 0.1% Tween-20 for 2 h, the membrane was infiltrated with the primary mouse anti-Syt-1 antibody (4 µg/mL, R&D Systems, Minneapolis, MN, USA) at 4 °C overnight. The membrane was washed in Tris-Buffered saline Tween-20 (TBST) 3 times and incubated in the goat anti-mouse secondary antibody (1:3000, Servicebio, WH, China) at 37 °C for 1 h. GAPDH (1:1000, Servicebio, WH, China) was used as the protein control. The antigen-antibody complex was reacted with a horseradish peroxidase substrate (Millipore, Burlington, MA, USA) and visualized using the ImageQuant LAS 4000 min CCD camera (GE Healthcare, Boston, MA, USA). The bands were analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA). Values of Syt-1 were represented as the optical density ratio of the target protein bands to the related GAPDH bands.
6
Western Blot Analysis of Renal Proteins
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The dissected renal tissues were homogenized in ice-cold lysis buffer, which included 50 mm Tris (pH 7.4), 1% Triton X-100, 150 mm NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitors. After being centrifuged at 12,000g for 15 minutes at 4°C, the supernatant was collected. Samples (50 μg per lane) were loaded and then separated on a sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk and incubated with the primary antibodies against programmed cell death protein 4 (PDCD4) (rabbit polyclonal 1:1,000; Novus, Littleton, CO), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) (rabbit monoclonal 1:1,000; Abcam), total protein kinase B (Akt) (rabbit monoclonal 1:1,000; Cell Signaling Technology, Danvers, MA), phospho-Akt (rabbit monoclonal 1:1,000; Cell Signaling Technology), B-cell lymphoma-2 (Bcl-2, rabbit monoclonal 1:1,000; Cell Signaling Technology), IκB-α and phospho-IκB-α (mouse monoclonal 1:1,000; Cell Signaling Technology) overnight at 4°C, then incubated with a horseradish peroxidase-conjugated secondary antibody, and developed by chemiluminescent Horseradish Peroxidase Substrate (Millipore, Billerica, MA). Results were normalized with glyceraldehyde-3-phosphate dehydrogenase or β-actin and expressed as ratios to control.
7
Western Blot Analysis of Cell Signaling
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Whole cell lysates were obtained by direct lysis of the cells using an ice-cold Mammalian Protein Extraction Reagent (M-PER, Pierce). Nuclear and cytoplasmic fractionations were performed using the Nuclear and Cytoplasmic Extraction Kit (Pierce). Protein (20 μg) was resolved by 10 % SDS-PAGE and electro-transferred onto a polyvinylidene difluoride membrane. Western blotting was performed according to standard methods, using anti-cleaved-PARP, anti-p53, anti-cytochrome c, anti-Akt, anti-phosphorylated-Akt (ser473), anti-FOXO3a, anti-p21, anti-p27 and anti-β-actin antibodies (Cell Signaling Technology). The membranes were developed using an enhanced chemiluminescence detection system, horseradish peroxidase substrate (Millipore) and an ImageQuant LAS-4000 Chemiluminescence and Fluorescence Imaging System (FujiFilm).
8
Profiling NF-κB and Phospho-Kinase Pathways
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SCC143 cells were grown to 90% confluence in 10-cm plates and subjected to serum starvation for 24 h. Cells were collected by centrifugation and washed once with PBS. The washed cell pellets were suspended in extraction lysis buffer and incubated for 20 min at 4 °C. The protein concentration was determined by the Pierce BCA protein assay kit (Pierce, Rockford, IL, USA), according to the manufacturer’s instructions. Screening for different proteins in cell lysates was performed with the Proteome Profiler Human NF-κB Pathway Array and Proteome Profiler Human Phospho-Kinase Pathway Array (R&D Systems, Minneapolis, MN, USA). Horseradish peroxidase substrate (Millipore Corporation, Burlington, VT, USA) was used to detect the protein signal and data were captured by exposure in ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA). For semi-quantitative analysis, ImageJ software version 1.52a (National Institutes of Health, Bethesda, MD, USA) was used.
9
Profiling Kinase Phosphorylation in CaSki Cells
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CaSki cells were grown to 90% confluence in 10 cm plates, subjected to serum starvation for 24 h and then treated with CSC (10 μg/mL) and equivalent DMSO concentration for 2 h. Cells were collected by centrifugation and washed once with PBS. The washed cell pellets were suspended in extraction lysis buffer and incubated with 20 min at 4°C. The protein concentration was determined using the Pierce BCA protein assay reagent according to the manufacture’s instruction. Screening for different proteins in cell lysates were performed with a Proteome profiler array kit (R&D Systems). For the parallel determination of the relative levels of phosphorylation of Mitogen-Activated Protein kinases and other serine/threonine kinases. The array allows to detect the relative phosphorylation of these kinases: Akt1, HSP27, p38 beta, Akt2, JNK1, p38 delta, Akt3, JNK2, p38 gamma, Akt pan, JNK3, p53, CREB, JNK pan, p70, S6K, ERK1, MKK3, RSK1, ERK2, MKK6, RSK2, GSK-3 alpha/beta, MSK2, TOR, GSK-3 beta, p38 alpha. Horseradish peroxidase substrate (Millipore Corporation, United States) was used to detect protein signal and data were captured by exposure to Fujifilm Light films. The analysis of Films was performed using the NIH ImageJ software.
10
Determination of NGF Isoforms by Western Blot
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The different forms of NGF in media and in cell lysate were determined by western blotting. Both media and lysate samples were prepared as described above, but 500 µl of media was concentrated in Amicon Ultra-0.5 centrifugal filter units (Ultracel-30 membrane, Millipore) by centrifugation at 13,000 r/min for 25 min. The reverse spin was performed immediately at 2000 r/min for 2 min.
Lysate and concentrated media were run on 10% Tris-HCl gels (Novex) and transferred to polyvinylidene fluoride membrane (Millipore). Blots were then probed with a monoclonal mCherry antibody (ThermoFisher Scientific 16D7, 1:1000), and β-actin (Abcam ab8226, 1:1000) was used as the loading control. Secondary antibodies were purchased from Dako, and signal was detected using the chemiluminescent horseradish peroxidase substrate (Millipore).
Lysate and concentrated media were run on 10% Tris-HCl gels (Novex) and transferred to polyvinylidene fluoride membrane (Millipore). Blots were then probed with a monoclonal mCherry antibody (ThermoFisher Scientific 16D7, 1:1000), and β-actin (Abcam ab8226, 1:1000) was used as the loading control. Secondary antibodies were purchased from Dako, and signal was detected using the chemiluminescent horseradish peroxidase substrate (Millipore).
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