For western blotting assay, protein samples of strains were prepared and extracted as described previously [96 (link)]. Proteins separated on the SDS-PAGE gel were transferred onto a polyvinylidene fluoride membrane with a Bio-Rad electroblotting apparatus. The polyclonal anti-Flag A9044 (Sigma, St. Louis, MO, USA), monoclonal anti-GFP ab32146 (Abcam, Cambridge, MA, USA) and monoclonal anti-mCherry ab125096 (Abcam, Cambridge, UK) antibodies were used at a 1:2000 to 1:10000 dilution for immunoblot assays. The samples were also detected with the monoclonal anti-GAPDH antibody EM1101 (Hangzhou HuaAn Biotechnology co., Ltd.) as a reference. Incubation with a secondary antibody and chemiluminescent detection were performed as described previously [97 (link)]. The experiment was repeated three times independently.
Electroblotting apparatus
Manufactured by Bio-Rad
Sourced in United States, Germany
The Electroblotting apparatus is a laboratory equipment used for the transfer of proteins from a gel to a membrane. It allows for the efficient and consistent transfer of biomolecules, enabling further analysis and detection.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (4 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Nitrocellulose membrane, by Cytiva (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bca kit, by Beyotime (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Anti gpnmb, by R&D Systems (2 mentions)
Odyssey image scanner, by GE Healthcare (2 mentions)
Gel imaging scanning system, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Anti gfp ab32146, by Abcam (1 mentions)
Anti mcherry ab125096, by Abcam (1 mentions)
Anti flag a9044, by Merck Group (1 mentions)
Em1101, by HuaAn Biotechnology (1 mentions)
Anti acetyl histone h3, by Merck Group (1 mentions)
Anti histone h3, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Odyssey v3, by LI COR (1 mentions)
39 protocols using electroblotting apparatus
1
Western Blotting Assay for Protein Detection
2
Histone Modification Analysis in Fungi
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Mycelia of each strain were collected from YES broth incubated at 28°C for 72 h. Samples were frozen in liquid nitrogen and ground to a fine powder for subsequent protein extractions. Approximately 100 mg ground powder was resuspended in 1 ml radio immunoprecipitation assay lysis buffer (RIPA, Beyotime, Shanghai, China), and whole proteins were extracted according to the manufacturer’s instruction. Equal amounts of proteins were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, USA) using an electroblotting apparatus (Bio-Rad, USA). Histone modifications were detected with the anti-acetyl-histone H3 (1:2,000 dilution; Millipore), anti-acetyl-histone H3K9 (1:2,000 dilution; PTM BioLabs, Hangzhou, China), anti-acetyl-histone H3K14 (1:2,000 dilution; PTM BioLabs), and anti-histone H3 (1:750 dilution; Abcam, UK) antibodies. A horseradish peroxidase-conjugated goat anti-rabbit antibody was used as the secondary antibody (1:10,000 dilution; Abgent, USA). The WesternBrightTM Quantum chemiluminescent HRP substrate was used (Advansta, USA), and chemiluminescence was detected using the Gene Imaging System (Syngene, Hong Kong, China).
3
Western Blot Protein Detection Protocol
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Total cell lysates were prepared in RIPA buffer (150 mM NaCl, 50 mM Tris—HCl pH 7.4, 2 mM EDTA, 0.5% deoxycholate, 1mM Na3VO4, 20 mM NaF, 0.5%Triton X-100), supplemented with protease inhibitor cocktail (Roche Applied Science) and phenylmethylsulfonyl fluoride (PMSF). Lysates were cleared by centrifugation at 4°C for 10 min at 10,000 × g. Equal amounts of protein were subjected to electrophoresis on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes (Whatman, Dassel, Germany) using an electroblotting apparatus (Bio-Rad Laboratories, Hercules, CA, USA). Primary antibodies used were the polyclonal anti-Gpnmb (R&D Systems) and anti-tubulin-alpha (Cedarlane Laboratories Limited). Matching secondary IRDye-conjugated antibodies (Westburg) were used for detection in an Odyssey V3.0 (LI-COR Inc.).
4
Western Blot Analysis of SH-SY5Y Cell Lysates
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Cells were washed twice with ice cold PBS, and total cell lysates were obtained by adding 50 or 100 mL of RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, containing fresh protease inhibitor cocktail) to the SH-SY5Y cells (1 × 106 cells/mL) cultured in six-well plates. The whole cell lysate (15 µg/lane) was subjected to 10% SDS-PAGE. Proteins were transferred to PVDF membranes and fixed using an electroblotting apparatus (Biorad, Hercules, CA, USA). After blocking the membranes in TBS containing 0.1% Tween-20 and 5% dry milk at room temperature for 1 h, they were incubated with primary antibodies (1:1000) overnight followed by conjugated secondary antibodies of horseradish peroxidase (1:10,000) incubation (Santa Cruz, CA, USA) at room temperature for 1 h. The optical densities of antibody-specific bands were then analyzed with an Image Analyzer LAS-3000 (Fuji, Japan).
5
Protein Extraction and Western Blot Analysis
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Approximately 200 mg of finely ground mycelia was resuspended in 1 mL of lysis buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF]) and 10 μL of protease inhibitor cocktail (Sangon, Shanghai, China). Proteins were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane with a Bio-Rad electroblotting apparatus. Immunoblot analysis was performed with an anti-GFP primary antibody (1:5,000, Sigma-Aldrich, United States) and an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10,000, Sigma-Aldrich, United States). Luminescence signals were detected using Pierce ECL western blotting substrate (Thermo Fisher Scientific, Hillsboro, OR, USA) with a ChemiDoc XRS + system (Bio-Rad Laboratories, Hercules, CA, USA).
6
Western Blot Immunodetection Procedure
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For western blot analysis, the proteins separated on SDS-PAGE gels were transferred onto a polyvinylidene fluoride membrane with a BioRad electroblotting apparatus as described [40 (link)]. The signals on the blots were detected with anti-p44/42 MAP kinase antibody (Cell Signaling Technology, MA) or anti-GFP antibody (Thermo Fisher Scientific, USA) under the ECL Supersignal system (Pierce, Rockford, IL).
7
Amygdalar Protein Expression Quantification
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The protein levels of CRFR1, pCREB, and BDNF in the amygdala were detected of four rats from five groups each. After behavior experiments, the brain tissue was dissected and stored at −80°C until analysis. The procedures for processing the brain tissue and Western blotting analysis were both conducted as described previously [24 (link), 25 (link)]. Denatured samples were electrophoresed by using the sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel, and then transferred to a PVDF membrane by using an electroblotting apparatus (Bio-Rad, Hercules, CA, USA). After nonspecific blocking, the membrane was incubated overnight at 4°C with a primary antibody (mouse anti-β-actin monoclonal antibody diluted to 1 : 1000, or goat anti-CRF1R polyclonal antibody diluted to 1 : 500). After wash, the membranes were incubated with secondary antibody (goat anti-mouse antibody diluted to 1 : 10000) for 1 h. Protein bands were examined at 800 nm wavelength by using a fluorescence scanner of Odyssey Infrared Imaging System.
8
Western Blot Analysis of Osteogenic Markers
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Total proteins were extracted from cells by using lysis buffer (Beyotime, Shanghai, China), according to the manufacturer's instructions. Proteins from each sample were separated using 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes using an electroblotting apparatus (Bio-Rad, Hercules, CA, USA). After blocking in 5% nonfat milk/TBS-Tween 20 (TBST) solution at room temperature for 1 h, membranes were then incubated with primary antibodies specific for Runx2 (1 : 1000), PPARγ (1 : 1000), Collagen I (1 : 1000), C/EBPα (1 : 1000), p-Akt (1 : 1000), Akt (1 : 1000), GSK-3β (1 : 1000), β-catenin (1 : 1000), and β-actin(1 : 2000) at 4°C overnight. After washing with TBST, blots were then incubated at room temperature for 1 h with HRP-conjugated secondary antibodies diluted to 1 : 5000 in 5% nonfat milk/TBST. Protein bands were visualized using ECL reagents according to the manufacturer's instructions. The intensity values of each phosphorylated kinase were normalized to the corresponding total protein bands. Unless otherwise stated, β-actin was used as an internal control.
9
Western Blot Protein Analysis
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Samples were separated by SDS-PAGE in 10–16% gels and stained with 0.05% coomassie brilliant blue or transferred to a poly-vinylidene difuoride (PVDF) membrane (Millipore) using a electroblotting apparatus (Bio-Rad Laboratories, California) at 200 mA for 1 h. The membrane was blocked with 5% skim milk for 1.5 h at room temperature. Then, the membrane was incubated with primary antibodies against for 24 h at 4 °C. After washing, the membrane was incubated with HRP conjugated second antibody for 1 h. The target proteins were visualized and imaged by the Gel Doc XR System (Bio-Rad, Hercules, CA).
10
Western Blot Analysis of Protein Expression
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Lysates of the cells were prepared using a radioimmunoprecipitation assay buffer mixed with phenyl methylsulfonyl fluoride (Beyotime Institute of Biotechnology). The lysates were centrifuged for 10 min at 12,000 x g at a temperature of 4˚C. Concentrations of the proteins were determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). The proteins (20 µg samples/lane) were separated by SDS PAGE on 12% gels and subsequently electro-transferred using electro-blotting apparatus (Bio Rad Laboratories, Inc.) onto nitrocellulose membranes. The membranes were blocked with tris-buffered saline with 1/1,000 tween containing 5% non-fat milk for 1 h at room temperature. Subsequently the membranes were incubated overnight with antibodies against RbAp48 (cat. no. ab79416; 1:1,000; Abcam), NF-κB (cat. no. 545380-34-5; 1:1,000; Merck KGaA) and GAPDH (cat. no. D16H11; 1:1,000; Cell Signaling Technology, Inc.) at 4˚C. After washing, membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.). After incubation, membrane washing was performed with TBS and Tween-20 followed by enhanced chemiluminescence reagent (EMD Millipore) treatment. For analysis, Gel Pro Analyzer software version 4.0 (Media Cybernetics, Inc.) was used.
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