Briefly, 10 μg of total protein extracts was loaded onto SDS–PAGE. After electrophoresis, proteins were transferred onto a Protran BA85 nitrocellulose membrane (Whatman, Perkin Elmer). Blots were hybridized with primary antibodies and horseradish peroxidase–conjugated secondary antibodies (170-65-16 and 170-6515; Bio-Rad, Marnes-la-Coquette, France) and revealed with enhanced chemiluminescence (ECL) blotting detection reagents (Amersham, Velizy-Villacoublay, France) or Clarity Western ECL substrate (Bio-Rad). Western blot imaging was performed with Chemidoc MP (Bio-Rad) based on charge-coupled device detection technology. Image capture and analysis of Western blots were controlled by Image Lab software. Quantification of Western blots was performed by ImageJ software (National Institutes of Health, Bethesda, MD).
Ecl blotting detection reagent
Manufactured by Cytiva
Sourced in United Kingdom
ECL blotting detection reagents are chemiluminescent reagents used for the detection of proteins in Western blot analysis. These reagents produce a light signal when exposed to the horseradish peroxidase (HRP) enzyme, which is commonly used to label target proteins in Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Protran, by Cytiva (2 mentions)
Horseradish peroxidase coupled anti rabbit or anti mouse antibodies, by Cell Signaling Technology (2 mentions)
Complete mini, by Roche (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Autoradiography film, by Fujifilm (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Non fat milk, by Bio-Rad (2 mentions)
Ge 130pb, by Cole-Parmer (2 mentions)
Kaleidoscope molecular weight markers, by Bio-Rad (2 mentions)
Whatman, by Cytiva (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Skimmed milk, by BD (1 mentions)
Gapdh, by Abcam (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Protease inhibitors cocktail, by Promega (1 mentions)
Ab290, by Abcam (1 mentions)
Cxcr4, by Cell Signaling Technology (1 mentions)
Sdf 1, by Abcam (1 mentions)
8 protocols using ecl blotting detection reagent
1
Western Blot Protein Detection Protocol
2
Western Blot Protein Analysis Protocol
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Tissue samples or cells were lysed in radio-immunoprecipitation assay lysis buffer containing 150 mmol/L NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mmol/L Tris (pH8) and a protease inhibitors cocktail (Promega; Fitchburg, WI, USA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Membranes were blocked using 5% skimmed milk (BD Biosciences) in Tris-buffered saline with Tween 20 and then probed with specific primary antibodies against the following proteins: SDF-1 (1: 1000, ab9797; Abcam, Cambridge, MA, USA); GFP (1: 1000, ab290; Abcam); CXCR4 (1: 1000, #2024; Cell Signaling, Danvers, MA, USA); signal transducer and activator of transcription 3 (STAT3; 1: 2000, #4904; Cell Signaling, Danvers, MA, USA); p-STAT3 (1: 2000, #9145; Cell Signaling); GAPDH (1: 10000, #5632–1; Epitomics, Burlingame, CA, USA). Membranes were then subsequently incubated with horseradish peroxidase-coupled anti-rabbit or anti-mouse antibodies (both 1: 2500; Cell Signaling). Specific bands were visualized using ECL Blotting Detection Reagents (Amersham Biosciences; Little Chalfont, Buckinghamshire, UK).
3
Western Blot Analysis of Autophagy Marker LC3-B
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Cells were harvested 24 hours after transfection and lysed in 50 mmol/L Tris-HCl, pH 7.5; 150 mmol/L NaCl; 1% NP-40, containing Complete Mini protease inhibitor mixture (Roche Diagnostics). Protein extracts were electrophoresed on 4% to 12% linear gradient Bis-Tris ready gels (Invitrogen) and transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore). Membranes were probed with antibodies specific for LC3-B (1:1,000, 2775S; Cell Signaling, Inc.) and actin (1:2,500, CB1001; Calbiochem), and subsequently with horseradish peroxidase-coupled anti-rabbit or anti-mouse antibodies (both 1:2,500; Cell Signaling Inc.). Specific bands were visualized with ECL Blotting Detection Reagents (Amersham Biosciences) and quantified with Image J software.
4
Western Blot Analysis of Tight Junction Proteins
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Radio-immunoprecipitation assay buffer consisting of 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris (pH 8), and protease inhibitor cocktail (Promega, Fitchburg, WI) was used to lyse cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to separate the proteins, which were then transferred to polyvinylidene fluoride membranes using the Trans-Blot System (Bio-Rad, CA). The membranes were incubated with specific primary antibodies against ZO-1 (Abcam, ab96587), claudin 3 (Abcam, ab199635), occludin (Abcam, ab167161), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, ab181602) at 4°C overnight after the membranes were blocked with 5% (w/v) skim milk (BD Biosciences, San Jose, CA) in Tris-buffered saline with 0.1% (v/v) Tween 20 (TBST) at room temperature for 1 h. The membranes were then washed with TBST three times and incubated with a specific secondary antibody (HRP goat anti-rabbit IgG, BA1054, Boster, China) at room temperature for 1 h. Enhanced chemiluminescence (ECL) Blotting Detection Reagents (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) were used to visualize the specific bands. GAPDH served as a control for the western blot analysis.
5
Western Blot Protein Analysis
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Total cell lysates were mixed with an equal volume of RIPA buffer, then heated for 10 min, analyzed using 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). Blots were reacted with slow shaking in blocking buffer (5% nonfat milk in PBS) for 1 h, probed with protein-specific primary antibodies at 4 °C overnight, washed with Tris Buffered Saline Buffer with Tween 20 (TBST), and then incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature. Finally, antigen-antibody complexes were developed using enhanced chemiluminescence (ECL) blotting detection reagents (Amersham Biosciences, Mountain View, CA, USA). Relative image density was analyzed using a Fuji LAS-3000 imaging system (FUJFILM, Tokyo, Japan).
6
Immunoblotting Analysis of Protein Extracts
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Cells or brain tissues were homogenized with an ultrasonicator (GE 130PB, Cole-Parmer, Vermon Hills, IL, USA) in 0.05 M HCl-Tris, pH 9.0 buffer, in the presence of a protease inhibitor cocktail (Mini-complete, Roche, Mannheim, Germany). The extracts were centrifuged (12,500 rpm, 15 min) and the supernatants collected. Total proteins were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and 50 µg of protein/lane was separated in 12.5% SDS-PAGE slabs, and then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) as previously described [53 (link)]. For immunoblotting, membranes were blocked with 5% non-fat milk (Bio-Rad) in TBS for 2 h. Then, membranes were incubated overnight at RT with the corresponding primary antibodies (Table 1 ) in TTBS. After washing the membranes with TTBS (3 × 10 min), they were incubated for 2 h with the corresponding HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using ECL blotting detection reagent (Amersham-Pharmacia, Buckinghamshire, UK) on autoradiography film (Fujifilm, Tokyo, Japan). Kaleidoscope molecular weight markers (Bio-Rad) were used as reference for molecular mass determination.
7
Hippocampal Protein Extraction and Western Blot
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Hippocampal cell cultures (4 wells per experimental group) were homogenized with an ultrasonicator (GE 130PB, Cole-Parmer, Vermon Hills, IL, USA) in 0.05 M HCl-Tris, pH 9.0 buffer, in the presence of a protease inhibitor cocktail (Mini-complete, Roche, Mannheim, Germany). The extracts were centrifuged (12,500 rpm, 15 min), and the supernatants were collected. Total proteins were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and 50 μg of protein/lane was separated in 12.5% SDS-PAGE slabs and then transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) as previously described [24 (link)]. For immunoblotting, membranes were blocked with 5% nonfat milk (Bio-Rad) in TBS for 2 h. Then, membranes were incubated overnight at RT with the corresponding primary antibodies (Table 1 ) in TTBS. After washing the membranes with TTBS (3 × 10 min), they were incubated for 2 h with the corresponding HRP-conjugated secondary antibodies. Immunoreactive bands were visualized using the ECL blotting detection reagent (Amersham-Pharmacia, Buckinghamshire, UK) on autoradiography film (Fujifilm, Tokyo, Japan). Kaleidoscope molecular weight markers (Bio-Rad) were used as reference for molecular mass determination.
8
Western Blot Protein Analysis Protocol
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Briefly, 10 µg of total protein extracts were separated by SDS-PAGE on acrylamide/bisacrylamide (Euromedex) or 3–15% gradient Tris-Acetate gels, in Tris-Glycin 1× 0.1% SDS buffer or in Tris-Acetate SDS Running Buffer [82 (link)]. After electrophoresis, proteins were transferred onto Protran BA85 nitrocellulose (Perkin Elmer, Waltham, MA, USA) or immobilon-P (Millipore) membranes and blots were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Revelation was performed with ECL detection reagents (Clarity Western ECL substrate from Bio-Rad or ECL blotting detection reagent from Amersham). Western blot imaging was performed with Chemidoc MP (Bio-Rad) based on charge-coupled device detection technology. Image capture and analysis of Western-blot were processed by ImageLab 4.0 software and quantification by Image J software (NIH).
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