Control and experimental cells were collected and lysed in radio‐immunoprecipitation assay (RIPA) buffer (cat. no. 20–188; EMD Millipore). After centrifugation (16,000 × g; 4°C) for 20 min, cellular protein was obtained from the supernatant layer. The protein concentration was determined with a protein assay kit (cat. no. 23200; Thermo Fischer Scientific, Inc.). Equal quantities (30 μg) of protein were separated in a 13.3% SDS gel and transferred onto polyvinylidene difluoride membranes (EMD Millipore). After washing in phosphate‐buffered saline (PBS), the membranes were blocked with 5% nonfat milk for 2 hr at room temperature. After washing with PBS, the membranes were treated with primary antibodies for 4 hr. Next, the membranes were washed with PBS and treated with anti‐rabbit HRP‐conjugated secondary antibodies for 1 hr at room temperature. The immunolabeled proteins were treated with Western Lightning® Chemiluminescence Plus reagent (PerkinElmer, Inc.) and determined with a Luminescence Image Analysis system (LAS‐4000, FUJIFILM Electronic Materials Taiwan Co., Ltd.).
Western lightning chemiluminescence reagent plus
Manufactured by PerkinElmer
Sourced in United States, Germany
The Western Lightning Chemiluminescence Reagent Plus is a laboratory reagent designed for the detection of proteins in Western blotting procedures. It generates a chemiluminescent signal in the presence of target proteins, allowing for visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Las4000mini ccd camera apparatus, by GE Healthcare (4 mentions)
Anti flag m2, by Merck Group (4 mentions)
Nitrocellulose membrane, by Merck Group (4 mentions)
Fuji las 4000, by Fujifilm (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Bradford protein assay, by Bio-Rad (3 mentions)
Immobilon p, by Merck Group (3 mentions)
Anti actin, by Merck Group (3 mentions)
Immobilon p transfer membrane, by Merck Group (2 mentions)
Protein assay kit, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
112 protocols using western lightning chemiluminescence reagent plus
1
Western Blotting Protein Quantification
2
Western Blot Protein Analysis Protocol
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Cells were treated with the RIPA buffer (EMD Millipore, Billerica, MA, USA). Cellular proteins were collected from the supernatant with centrifugation (16,000 × g) at 4 °C for 20 min. The protein concentration was determined by using a protein assay kit (Thermo Fischer Scientific, Inc., Waltham, MA, USA). Equal quantities (40 μg) of protein were separated by 13.3% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were treated with 5% non-fat milk at room temperature for 2 hours and washed with PBS buffer for 15 minutes (three times). The PVDF membranes were treated with primary antibodies at room temperature for 4 hours. Next, PVDF membranes were washed three times with PBS buffer for 15 minutes, then the membranes were treated with anti-rabbit HRP-conjugated secondary antibodies at room temperature for 1 hour. Finally, the membranes were treated with Western Lightning® Chemiluminescence Plus reagent (PerkinElmer, Inc., Waltham, MA, USA) and observed with a Luminescence Image Analysis system (LAS-4000, FUJIFILM Electronic Materials Taiwan Co., Ltd., Tainan, Taiwan).
3
SDS-PAGE Immunoblotting Analysis
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Harvested proteins were electrophoretically separated by 12% (w/v) SDS-PAGE in non-reducing conditions and transferred to Immobilon-P Transfer Membrane (Millipore, Darmstadt, Germany). After blocking with 5% milk-TBS-0.1% Tween 20 buffer at RT for 1 h, the membrane was stained with the indicated primary and secondary antibodies at RT for 1 h, as done previously in this lab. After final washing, the membrane was treated with Western Lightning Chemiluminescence Plus Reagent (PerkinElmer, Waltham, MA, USA) and signals were detected by FUJI X-ray film.
4
Protein Extraction and Western Blot Analysis
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Control and experimental cells were lysed in the radio-immunoprecipitation assay buffer (cat. no. 20-188; EMD Millipore, Billerica, MA, USA). Cellular proteins were obtained from the supernatant with centrifugation (16,000 × g; 4 ºC) for 20 min. The protein concentration was determined using a protein assay kit (cat. no. 23200; Thermo Fischer Scientific, Inc., Waltham, MA, USA). Equal quantities (40 μg) of protein were separated by SDS-PAGE (13.3% gels, 80 volts) and transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were treated with 5% non-fat milk at room temperature for 2 hours and washed with PBS buffer for 15 minutes (three times). Next membranes were incubated with primary antibodies at room temperature for 4 hours. After membranes were washed with PBS buffer for 15 minutes, the membranes were treated with anti-rabbit HRP-conjugated secondary antibodies at room temperature for 1 hour. Finally, the membranes were treated with Western Lightning® Chemiluminescence Plus reagent (PerkinElmer, Inc., Waltham, MA, USA) and observed with a Luminescence Image Analysis system (LAS-4000, FUJIFILM Electronic Materials Taiwan Co., Ltd., Tainan, Taiwan).
5
Dengue Virus E Protein Detection
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Protein harvested from C6/36 cells that were either infected by DENV2 or not was boiled for 3 min, then separated by electrophoresis on 12% (w/v) sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) in nonreducing conditions. It was subsequently transferred onto an Immobilon-P Transfer Membrane (Millipore, Darmstadt, Germany). After blocking with 5% milk-TBS-0.1% Tween 20 buffer at RT for 1 h, the membrane was incubated with the indicated primary and secondary antibodies at RT for 1 h as the method done previously in this lab [29 (link)]. Specific primary antibodies included the 4G2 monoclonal antibody (a kind gift of Dr. Guey-Chuen Perng, National Cheng Kung University, Taiwan) for the dengue E protein and anti-actin mouse monoclonal antibody clone C4 (Merck Millipore, Burlington, MA, USA). Secondary antibodies were goat anti-rabbit or anti-mouse IgG antibodies, depending on the primary antibody used in the experiment. After the final wash, the membrane was treated with a Western Lightning Chemiluminescence Plus Reagent (PerkinElmer, Waltham, MA, USA), from which signals were detected on a FUJI X-ray film.
6
Immunoblotting of Bacterial RecA Protein
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Cells from 1 ml culture were pelleted, resuspended in 100 μl SDS lysis buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, 5% 2-mercaptoethanol, 50 mM dithiothreitol), and boiled for 5 min. After boiling, suspension was pelleted and 15 μl of supernatant was run on a 10% acrylamide (37.5:1 acrylamide:bis) SDS-PAGE gel by standard methods. Protein was transferred to Immobilon-P membrane (Millipore) using a BioRad Mini-PROTEAN 3 Trans-Blot electrophoretic transfer cell. Blots were incubated at 4°C for 12–14 h with monoclonal mouse anti-RecA antibody diluted to 0.11 ng/ml in blocking buffer. Secondary labeling was done using horseradish peroxidase conjugated sheep anti-mouse antibody (GE Healthcare) diluted 1:1000 in blocking buffer. Blots were treated with Western Lightning Plus chemiluminescence reagent (Perkin Elmer) and imaged using the Alpha Innotech FluorChem SP imaging system.
7
Fly Head Protein Extraction and Western Blot
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Fly heads were obtained from 3 independent crosses through flash freezing with liquid nitrogen, followed by vortexing and sieving to separate heads from bodies. Homogenization of samples was obtained using a motorized pestle and 100 µL T-PER (Thermo Fischer Scientific) containing protease inhibitors (Sigma). 20/30 µg of protein was subjected to standard SDS-PAGE and Western blotting with HFP-conjugated secondary antibodies. The HRP-signal was visualized after incubation with the West Pico Plus chemiluminescent reagent (Thermo Fischer Scientific)) or the Western Lightning™ Plus Chemiluminescence Reagent (Perkin Elmer) using the iBright imaging system (Thermo Fisher Scientific). The following antibodies were used: rabbit anti-GAPDH [1:2000 (Abcam)], rabbit anti-DNAJC6 [1:1000 (Pierce)], rabbit α-Synj [1:2000;19 (link)], rabbit anti-GAPDH [1:10000 (Invitrogen)] and HRP-conjugated anti-rabbit seconday antibodies [1:5000 (Jackson ImmunoResearch)].
8
Western Blot Analysis of ACSLs
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A 10-µl aliquot of cell lysate was subjected to SDS/PAGE using a 10% (w/v) gel under reducing conditions. The separated proteins were electroblotted on to nitrocellulose membranes (GE Healthcare Bioscience, Piscataway, NJ, U.S.A.) with a bath-type blotter. After blocking for 1 h with 5% (w/v) skimmed milk in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (TPBS), the membranes were probed for 1 h with the respective antibodies (1:1000 for ACSL1, ACSL3, ACSL5, and ACSL6, 1:5000 for ACSL4, and 1:10000 for β-actin), followed by incubation with horseradish peroxidase-conjugated anti-rabbit (1:3000 for ACSL1, ACSL4, ACSL5, and ACSL6) and anti-mouse (1:3000 for ACSL3 and β-actin) IgG. After washing, the membranes were visualised using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Wellesley, MA, U.S.A.).
9
MYC Protein Detection by Western Blot
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Cells were collected in chilled Dulbecco’s Phosphate Buffered Saline (Hyclone) and lysed with sonication in high salt lysis buffer (50 mM Tris, pH 8; 500 mM NaCl; 1% Triton X-100) with protease inhibitors (Complete Mini Protease Inhibitor; Roche). Protein was quantified and separated by SDS-PAGE in a 13% poly-acrylamide gel then transferred to PVDF. Membranes were blocked for at least 1 h in 5% skim milk powder in PBS containing 0.1% Tween-20. Membranes were incubated for at least 2 h in primary antibodies: α-MYC (1:500 monoclonal 9E10 in blocking solution) or anti-β-actin (Genetex at 1:2000) and 1 h in secondary (1:10,000 HRP-conjugated goat-α-mouse, Bio-Rad). Immunoreactive bands were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences) and imaged with a Bio-Rad molecular Imager Chemi-Doc XRS+ and ImageLab software (Bio-Rad).
10
Western Blot Analysis of IFNAR1
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Cells were lysed in modified RIPA buffer (50 mM Tris-HCl pH 8, 200 mM NaCl, 1% NP40, 0.5% DOC, 0.05% SDS, 2mM EDTA) with 1 mM Na3VO4 and a cocktail of antiproteases (Roche). A total of 30 μg of proteins was separated by SDS-PAGE and analyzed by western blot. Membranes were cut horizontally according to molecular size markers, and stripes were incubated with different Abs. Immunoblots were analyzed by ECL with the ECL Western blotting Reagent (Pierce) or the more sensitive Western Lightning Chemiluminescence Reagent Plus (PerkinElmer) and bands were quantified with Fuji LAS-4000. For reprobing, blots were stripped in 0.2 M glycine (pH 2.5) for 30 min at room temperature. The following Abs were used: TYK2 mAb T10-2 and anti-GST (Hybridolab, Institut Pasteur); anti-IFNAR1 (64G12 mAbs) [60 (link)]; anti-STAT2-phospho-Y689 (R&D); Abs to STAT1, STAT2, STAT3, and STAT1-P-Y701, STAT3-P-Y705, and TYK2–P-YY1054/55 (Cell Signaling Technology, Beverly, MA).
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