Cells were collected after transfection, washed with DPBS (Corning), and lysed in ice-cold lysis buffer (100 mM NaCl, 10 mM Tris, pH 7.5, 1 mM EDTA, 0.5% Nonidet P-40, protease and phosphatase inhibitor cocktail [Roche]). Cell lysates were quantified by the Bradford assay (Bio-Rad) and 20 µg of lysates were denatured in Laemmli sample buffer and incubated for 5 min at 95°C. The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% nonfat milk in Tris-buffered saline pH 7.4 and 0.5% Tween 20 (TBST) and then incubated with primary antibodies. After washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Rockland Immunochemicals) and detected using Western Lightning Plus-ECL reagent (Perkin-Elmer). Signal intensity and densitometry analyses were conducted using ImageJ (NIH).
Western lightning plus ecl reagent
Manufactured by PerkinElmer
Sourced in United States, United Kingdom
The Western Lightning Plus-ECL reagent is a chemiluminescent detection system used in Western blot analysis. It is designed to enable sensitive and reliable detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (13 mentions)
Chemocam imager 3, by Intas (6 mentions)
Genetools software, by Syngene (6 mentions)
Genegnome xrq system, by Syngene (6 mentions)
Horseradish peroxidase conjugated secondary antibody, by Rockland Immunochemicals (4 mentions)
Ipvh00010, by Merck Group (4 mentions)
P1260, by Solarbio (4 mentions)
Bl507a, by Biosharp (4 mentions)
Colorimetric assay, by Bio-Rad (4 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse ab, by PerkinElmer (4 mentions)
Bradford assay, by Bio-Rad (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Nupage gel, by Thermo Fisher Scientific (3 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ripa lysis buffer, by Cell Signaling Technology (3 mentions)
Quantum protein assay, by Euroclone (3 mentions)
109 protocols using western lightning plus ecl reagent
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Protein Expression
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Cells were lysed in 2x Laemmli buffer (200 mM Tris-HCl [pH8.8], 5 mM EDTA, 0.1% (w/v) Bromophenol Blue, 10% (w/v) Sucrose, 3% (w/v) SDS, 2% β-Mercaptoethanol) and heated to 98°C for 10 minutes. Proteins were separated by SDS-PAGE and blotted onto a PVDF membrane by either semi-dry or wet-blot (Bio-Rad, Germany). Membranes were blocked with 5% milk in PBS containing 0.1% Tween 20 for one hour at room temperature or overnight at 4°C. Membranes were incubated with primary antibodies overnight at 4°C or for 90 minutes at room temperature. Secondary antibodies tagged with horseradish peroxidase were incubated for one hour at room temperature. After washing with PBS containing 0.1% Tween 20, proteins were visualized by using the Western Lightning Plus-ECL reagent (PerkinElmer, USA) and an INTAS Advanced Fluorescence and ECL imager (INTAS, Germany) or a Curix 60 Developer device (AGFA, Germany). The Lab Image 1D software package (Kapelan BioImaging Solutions, Germany) was used for quantification of specific signals that were normalized to β-actin or GAPDH or wild type MAVS expression as specified in the results section.
3
Bocaviral Capsid Protein Detection
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Western blot analysis was performed as previously described.94 (link) Briefly, 10 μL from each iodixanol-purified virus stock was mixed with an equal volume of 2× SDS sample loading buffer and boiled for 5 min at 95°C. Then, the samples were separated on 8% SDS-PAGE gels and transferred to a nitrocellulose membrane (NeoLab, Heidelberg, Germany) via semi-dry transfer. Membranes were blocked with 5% milk (Roth, Karlsruhe, Germany) for 1 h at RT and incubated overnight with an anti-VP polyclonal primary rabbit antibody (1:1,000 dilution) recognizing the three bocaviral capsid proteins VP1, VP2, and VP3 (kind gift from Maria Söderlund-Venermo, University of Helsinki). For detection, a horseradish peroxidase-conjugated secondary donkey anti-rabbit antibody (GE Healthcare, Chicago, IL, USA; NA934V) was used at a 1:10,000 dilution. To visualize protein bands, Western Lightning Plus-ECL reagent (PerkinElmer, Waltham, MA, USA) was used, and the emitted signal was detected with a chemiluminescence imager (Intas ChemoStar, Göttingen, Germany).
4
Western Blot Analysis of Whole-Cell Extracts
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For Western blotting analysis, whole-cell extracts were prepared by scrapping the cells growing as a monolayer into RIPA buffer (1% NP-40, 50 mM Tris-HCl pH 8.0, supplemented with protease and phosphatase inhibitors). The cellular lysates were incubated for 1 h at 4 °C and then clarified by centrifugation for 30 min at 13,000 rpm at 4 °C. Protein concentrations were measured by the Bradford assay. The samples containing 30 μg of protein were resolved on 4–12% Bis-Tris or 3–8% Tris-acetate NuPAGE gels (Invitrogen, Carlsbad, CA, USA), transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), probed with specific antibodies and visualized by enhanced chemiluminescence (Western Lightning Plus-ECL reagent, Perkin Elmer, Waltham, MA, USA). Densitometric analysis of Western blotting images was performed by using the NIH ImageJ software (Bethesda, MD, USA), which was downloaded from the NIH website (http://rsbweb.nih.gov/ij/download.html ).
5
Profiling Human Soluble Receptors in Cell Lines
6
Western Blot Protein Detection Protocol
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Cells were collected and lysed with radioimmunoprecipitation assay lysis buffer (P0013; Beyotime) containing phenylmethylsulfonyl fluoride (BL507A; Biosharp) and phosphatase inhibitors (P1260; Solarbio) on ice for 30 min. The components in the supernatant (denatured at 100°C in sample buffer) were separated using SDS-PAGE and transferred to 0.45-μm or 0.22-μm polyvinyl difluoride (PVDF) membranes (IPVH00010; Millipore). The PVDF membranes then were blocked with 5% nonfat milk for 1 h and incubated overnight with primary antibodies at 4°C. Subsequently, the PVDF membranes were washed with Tris-buffered saline-Tween 20 and incubated with the corresponding secondary antibody for 1 h. Finally, the proteins were detected with Western Lightning plus-ECL reagent (NEL104001EA; PerkinElmer) and detected with a Synoptics Syngene bioimaging instrument (R114075; Synoptics).
7
Western Blotting Protein Analysis
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Western blotting was performed as described previously.25 (link) Briefly, proteins were transferred to nitrocellulose membrane using a Trans-Blot Turbo Transfer System (BioRad, Montreal, QC, Canada), transfer was confirmed using Ponceau S stain (0.1% (w/v) in 5% (v/v) acetic acid) and the membranes were blocked in 5% skim milk in Tris-buffered saline with 0.1% (v/v) Tween-20 (TBS-T; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween-20) at room temperature for 2 hours. The membranes were incubated overnight at 4°C with the following primary antibodies diluted in 5% (w/v) skim milk in TBS-T: anti-poly-(ADP- ribose) polymerase (PARP) (1:3,000, 9542S; Cell Signaling Technology, Whitby, ON, Canada), anti-caspase 3 (1:500, 9662S; Cell Signaling Technology), anti-caspase 7 (1:1,000, 9492S; Cell Signaling Technology), anti-caspase 9 (1:1,000, 9502S; Cell Signaling Technology), and anti-β-actin (1:5,000, 8457L; Cell Signaling Technology). Membranes were washed three times with TBS-T, incubated with anti- rabbit secondary antibody (1:3,000, 7074S, Cell Signaling Technology), washed three times with TBS-T, incubated with Western Lightning Plus-ECL reagent (PerkinElmer Inc., Waltham, MA, USA) for 2 minutes, and developed using the chemiluminescence setting on a ChemiDoc MP Imaging System (BioRad).
8
Western Blotting of Fusion Proteins
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After SDS-PAGE of the collected SEC fractions (10 μL per fraction) on a 4–15% acrylamide gradient gel, the proteins were transferred onto a PVDF membrane using a Trans-Blot Turbo semi-dry transfer system (Bio-Rad). In the cases where the cargo proteins were fusion constructs with N- or C-terminal parts of TccC3HVR, a custom-made anti-TccC3HVR rabbit polyclonal antibody (Cambridge Research Biochemicals) was used as the primary antibody at 1:1000 dilution. For Cdc42 without a TccC3HVR fusion, an anti-Cdc42 rabbit polyclonal antibody (Cell Signaling Technology, Cat. No. 2462) was used as the primary antibody at 1:1000 dilution. For TEV, an anti-TEV protease rabbit polyclonal antibody (Novus Biologicals, Cat. No. NBP1–97669) was used as the primary antibody at 1:500 dilution. An HRP-conjugated goat anti-rabbit antibody (Bio-Rad, Cat. No. 170–6515) was applied as the secondary antibody at 1:2000 dilution in all cases. Detection was performed with Western Lightning Plus ECL reagent (PerkinElmer, Cat. No. NEL104001EA) and imaged in a ChemiDoc MP imaging system (Bio-Rad).
9
Cytokine Profiling of ASC Secretome
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Transduced and Puromycin-selected ASCs were seeded in six-well plates at a density of 5 × 103 cells/cm2 and incubated overnight. On the next day, the supernatant was aspirated and replaced by 1 mL/well PM4 medium for 72 hours. Conditioned media were collected and cells were harvested to gain RNA and protein samples, respectively. Culture supernatants were stored immediately at −80°C until analysis. Corresponding supernatants of three different donors were equally mixed and analyzed by the Proteome Profiler Human Cytokine Array (RnDSystems, #ARY005B) as described in the manufacturer’s instructions. This array can detect 36 different cytokines. Development of the signal was achieved with the Western Lightning Plus ECL reagent (PerkinElmer) and membranes were exposed to a medical X-ray film (Super RX-N, FUJI) for up to 60 minutes. Quantification was performed with ImageJ software (version 1.47, National Institutes of Health).
10
Western Blotting of IVC Proteins
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