Protein samples were run on an SDS-PAGE gel alongside a pre-stained ladder (Invitrogen, Waltham, U.S.) and then transferred onto a nitrocellulose membrane (Bio Rad Laboratories, Hercules, U.S.) in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20% (v/v) methanol, pH 8.3) using a TE77 semi-dry transfer unit (Hoefer, Holliston, U.S.) at 200 V, 50 mA/gel for 1 h. The membrane was incubated by blocking solution (TBS-T plus 5% (w/v) skim milk powder) on a shaker at RT for at least 1 h to block nonspecific binding sites of the membrane. Following this step, the membrane was washed twice with TBS-T (20 mM Tris-Cl, 150 mM NaCl, pH 7.6, 0.1% (v/v) Tween-20) for 5 min. The membrane was probed with human sera from patients with invasive GAS infections and healthy donors at 1/100 dilution in probing solution (TBS-T plus 2.5% (w/v) skim milk powder) for 1 h at RT. The membrane was then washed three times with TBS-T for 5 min each and incubated with HRP-conjugated goat anti-human IgG (Abcam) in a probing buffer and incubated for 1 h followed by washing with TBS-T three times for 5 min. Western blots were developed with ECL detection reagent (Amersham Biosciences) and analyzed using a ChemiDocTM imaging system (BioRad).
Ecl detection reagent
Manufactured by Cytiva
Sourced in United Kingdom, United States, Sweden, Germany, Australia, Japan, Switzerland
The ECL detection reagent is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. It reacts with the enzyme-labeled secondary antibody to produce a luminescent signal, which can be captured using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (18 mentions)
Ripa lysis buffer, by Beyotime (8 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (8 mentions)
β actin, by Cell Signaling Technology (7 mentions)
Hybond, by Cytiva (7 mentions)
Protein assay kit, by Bio-Rad (6 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Bradford assay, by Bio-Rad (6 mentions)
Quantity one software, by Bio-Rad (5 mentions)
Gapdh, by Cell Signaling Technology (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Fusion fx7 system, by Avantor (5 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Nitrocellulose membrane, by Cytiva (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
Hyperfilm ecl, by Cytiva (5 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (4 mentions)
Amersham ecl, by Cytiva (4 mentions)
189 protocols using ecl detection reagent
1
Western Blot Analysis of GAS Infection
2
Western Blot Analysis of Cells
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The HDF cells or NHEK cells were seeded on either 6 well (1 × 105 cells / well) or 12 well plate (5 × 104 cells/well), and were cultured for 24 h. The CD extract was treated on the cells for an appropriate time and the cell lysates were harvested. The cell lysates were then separated by SDS-PAGE electrophoresis and the proteins were blotted onto a nitrocellulose membrane. After blocking with 0.5% bovine serum albumin solution containing 0.1% Tween-20, the membrane was incubated with primary antibodies overnight at 4 °C. Secondary antibodies conjugated with horse radish peroxidase were incubated for one hour under room temperature, and the protein quantity was measured using ECL detection reagent (Amersham Biosciences Corp., Little Chalfont, UK) using the ChemiDoc system (Alliance Mini HD 9, Uvitec, Cambridge, UK).
3
Proteomics analysis of SINT1 analogs
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LNCaP cells were treated for 24 h with 20 μm modified SINT1 (LPY19, -30, and -31) or the vehicle dimethyl sulfoxide (DMSO). 25 μm EPI-053 (a modified EPI-001 analog) was used as positive control (5 (link)). Proteins were extracted with lysis buffer and subjected to click chemistry conditions for 3 h at 25 °C in buffer containing 0.1% SDS, 5% t-butyl alcohol, 100 μm tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (Sigma), 1 mm tris(2-carboxyethyl)phosphine, 100 μm biotin-azide reagent, and 1 mm CuSO4. Samples were dialyzed in 50 mm HEPES, pH 8.0, 150 mm NaCl, 0.1% SDS, and 1% Triton X-100 to remove excess biotin-azide reagent. Biotinylated soluble receptors were captured with streptavidin-agarose resin. Protein-SINT1 complexes were separated by 10% SDS-PAGE and subjected to Western blotting analysis using AR-N20 (1:1000) and biotin (1:1000) antibodies. Proteins were visualized using ECL detection reagent (Amersham Biosciences).
4
Fractionation and Lipid Incorporation of Francisella tularensis
5
Western Blot Analysis of Transduced 293T Cells
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1×106 293T cells were transduced with 1×106 vp of each viral construct. After 48 hours, supernatant and cells were harvested for Western blot analysis. Cells were lysed in RIPA buffer (Biorad) and proteins were denatured with 2% SDS and reduced with 1% DTT before they were loaded into a 4–15% gradient Tris Glycene-SDS polyacrylamide gels (Bio-Rad), electrophoresed, and blotted onto PVDF membrane (Pierce). The membrane was blocked using 5% (w/v) dry milk and then probed with goat anti-mouse 4-1BBL or CD27L, or rat anti-mouse GITRL or BAFF (R&D Systems), followed by incubation with either anti-goat or anti-rat horseradish peroxidase-conjugated antibodies (Jackson Immunoresearch). The protein band was developed onto X-ray film using ECL detection reagent (Amersham).
6
Western Blot Analysis of Cell Signaling Proteins
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For total protein analysis, cells were harvested with lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.4% β-Mercaptoethanol, Proteinase-Inhibitor Cocktail, Roche, Mannheim, Germany). Clarified protein lysate was applied to a polyacrylamide gel and analyzed by western blotting [95 (link), 96 (link)]. To this end, 30-50 μg protein of whole cell lysate was incubated with primary antibody for Orai1 (1:1000, Millipore, Bedford, MA, USA, [22 (link)]), STIM1 (1:1000, cell signaling, Danvers, MA, USA, [97 (link)]), Akt (1:1000, cell signaling, Danvers, MA, USA, [93 (link)], Phospho-Akt (Thr308) (1:1000, cell signaling, Danvers, MA, USA, [93 (link)]and GAPDH (1:1000, cell signaling, Danvers, MA, USA, [98 (link)]). For detection secondary antibody conjugated with horseradish peroxidase (HRP) (1:2000, Cell Signaling, Danvers, MA, USA) was used. Antibody binding was identified with ECL detection reagent (Amersham, Freiburg, Germany). Bands were quantified with Quantity One Software (Biorad, München, Germany [22 (link)]). The appropriate band has been defined by using Orai1 overexpressing cells [22 (link), 24 (link)].
7
Western Blot Analysis of Hepatic PPARα
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Liver protein was extracted with lysis solution containing 1 mM EDTA, 1% Triton X-100, 150 mM HEPES, protease inhibitors, and phosphatase inhibitors (GenDEPOT, Barker, TX, USA). Equal amounts of protein were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 3% bovine serum albumin and then incubated overnight with primary antibodies at 4℃. Antibodies to peroxisome proliferator-activated receptor α (PPARα) and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). After incubation with primary antibodies, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) for 1 h at room temperature. The membranes were then treated with ECL detection reagent (Amersham Bioscience, Little Chalfont, UK). The relative band density was determined by densitometry with Image J software (National Institutes of Health, Bethesda, MD, USA).
8
Western Blotting for OXPHOS and GAPDH
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Western blotting was performed as previously described (17 (link),20 (link)). The cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA and 1% Triton X-100) containing a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). The total protein concentrations were measured by a BCA Protein Assay kit (Sigma-Aldrich; Merck KGaA). After heat-denaturing, equal quantities of proteins (20 µg) were separated by NuPAGE Novex 12% Bis-Tris Gel and electrophoresed in the XCell SureLock™ Mini-Cell (both from Invitrogen; Thermo Fisher Scientific, Inc.), and then transferred to polyvinylidene difluoride membranes and blocked with 5% non-fat milk in Tris-buffered saline (TBS) for 1.5 h at the room temperature. The membranes were incubated with primary antibodies against OXPHOS (ab110413; 1:1,000; Abcam, Cambridge, UK) or GAPDH (AB2302; 1:1,000; EMD Millipore, Billerica, MA, USA) overnight at 4°C. After washing three times with 1X TBS, the membranes were incubated with secondary antibodies [anti-mouse (AP181R) and anti-rabbit antibodies (AP187R); 1:10,333; EMD Millipore] for 1.5 h at room temperature. After washing steps, immunoreactive binding was detected with ECL detection reagent (Amersham Biosciences, Piscataway, NJ, USA) with MicroChemi 4.2. The band intensity was quantified using ImageJ 1.47 software.
9
His-tag Protein Detection Protocol
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The proteins were separated by SDS-PAGE and were blotted onto polyvinylidene difluoride (PVDF) membranes using an iBlot2 Gel Transfer Device (Life Technologies, Carlsbad, CA) for 7 minutes. The membranes were blocked in 5% w/v skimmed milk powder/TBS buffer at room temperature for 1 hour and then incubated with mouse anti-His antibody (GenScript) at a dilution of 1:5,000 for 1 hour. After incubation, the membrane was washed three times with Tris buffered saline with Tween-20 (TBST) buffer, HRP-conjugated Rabbit anti-strep tag II antibody (diluted 3/10,000 in TBST) (GenScript) at room temperature for 1 hour, followed by washing with TBS-T buffer 3× for 10 minutes each. The protein was detected and visualized with ECL detection reagent (Amersham, Piscataway, NJ) following the manufacturer’s recommendation.
10
Western Blot Analysis of HCC Proteins
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Proteins extracted from HCC fresh tissue or cells with various treatments were fractionated by SDS-PAGE, transferred to PVDF membrane, and incubated with a primary specific antibody for CBX1 (1:1000, Abcam, ab10478), Phospho-β-Catenin (Ser552) (1:1000, Cell Signaling Technology, #5651), HMGA2 (1:1000, Cell Signaling Technology, #8179) in 5% of non-fat milk, followed by a horse radish peroxidase (HRP)-conjugated anti-rabbit second antibody. ECL detection reagent (Amersham Life Science, Piscataway, NJ, USA) was used to show the results.
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