The protein expression of nonstimulated and stimulated cells was measured by Western blotting analysis. Cells were treated with various concentrations of EDG and DC under serumfree conditions, nonstimulated for FcεRI expression, and stimulated with CRA-1 (10 μg/mL) for PTK and MAPK expression. Whole cell lysates were extracted with a cell lysis buffer containing 20 mM Tris-Cl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, 2 mM EDTA, and a protease inhibitor cocktail (Roche, Penzberg, Germany). Equal amounts of protein were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was blocked with 5% skim milk in plain buffer (20 mM Tris pH 7.4 and 136 mM NaCl) at room temperature for 1 h, and incubated with primary antibodies overnight at 4 °C. The membrane was then incubated with 500 times diluted specific secondary horseradish peroxidase (HRP)-conjugated antibodies at room temperature for 1 h, and the immunoreactive bands were visualized using enhanced chemiluminescence detection reagents (Perkin Elmer, Waltham, MA, USA), in according to the manufacturer’s instructions.
Enhanced chemiluminescence detection reagents
Manufactured by PerkinElmer
Sourced in United States
Enhanced chemiluminescence detection reagents are a set of reagents used for the detection and visualization of proteins or other biological molecules in Western blotting or other immunoassay techniques. These reagents generate a luminescent signal in the presence of the target analyte, allowing for sensitive and quantitative detection.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Polytron type homogenizer, by Kinematica (1 mentions)
2 7 dichlorofluorescin diacetate dcfda, by Merck Group (1 mentions)
Antibiotics and antimycotic, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence detection reagent, by PerkinElmer (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Rpmi 1640 medium, by Cytiva (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Sds sample buffer, by Bio-Rad (1 mentions)
Iblot apparatus, by Thermo Fisher Scientific (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Sds polyacrylamide gel electrophoresis, by Fujifilm (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Cytoplasmic nuclear extraction kit, by Thermo Fisher Scientific (1 mentions)
Hybond p membrane, by GE Healthcare (1 mentions)
Hybond, by Cytiva (1 mentions)
7 protocols using enhanced chemiluminescence detection reagents
1
Western Blot Analysis of Protein Expression
2
Analyzing Tight Junction Proteins in Mouse Colon
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A segment of mouse colonic tissues (20 mg) was homogenized in 600 μL lysis buffer with protease and phosphatase inhibitors (1% [w/v] sodium dodecyl sulfate, 1% [v/v] Triton X-100, 1% [w/v] sodium deoxycholate, and 30 mmol/L 2-amino-2-(hydroxymethyl)-1,3-propanediol trimethylolaminomethane) using a Polytron-type homogenizer (Kinematica AG, Lucerne, Switzerland). The supernatant obtained after centrifugation was subjected to immunoblot analyses of TJ proteins, ZO-1, ZO-2, occludin, claudin-3, claudin-4, and claudin-7 using specific antibodies in combination with horseradish peroxidase-conjugated anti-rabbit IgG antibodies. Blots were developed using enhanced chemiluminescence detection reagents (Perkin Elmer Life Sciences, Waltham, MA, USA).
3
Biochemical Analysis of Cell Signaling
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RPMI-1640 medium and fetal bovine serum (FBS) were purchased from HyClone Laboratories (Logan, UT, USA). CRA-1 was acquired from Kyokuto (Tokyo, Japan). Antibiotics and antimycotics were purchased from Gibco BRL (Gaithersburg, MD, USA). Protease inhibitor cocktail was obtained from Roche Diagnostics GmbH (Penzberg, Germany). β-Actin, anti-phosphorylated Syk, Lyn, and NF-κB, and horseradish peroxidase (HRP)-conjugated secondary antibody were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Chemiluminescence detection reagents were acquired from Perkin Elmer (Waltham, MA, USA), and polyvinylidene difluoride (PVDF) membrane was purchased from Millipore (Bedford, MA, USA). 2′7′-dichlorofluorescin-diacetate (DCF-DA) was obtained from Sigma Chemicals (St. Louis, MO, USA). Protease inhibitor cocktail was purchased from Roche (Penzberg, Germany). Enhanced Chemiluminescence detection reagents were procured from Perkin Elmer.
4
Western Blot Protein Quantification
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Cell extracts were prepared as described previously [13 (link)]. Equal amounts of protein were separated using SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and blocked with 5% nonfat milk and 1% Tween-20 in a Tris-buffered solution. Membranes were probed with the primary antibody overnight. After incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, blots were detected by enhanced chemiluminescence detection reagents (PerkinElmer, Waltham, MA, USA) and exposed to X-ray film (Super HR, Fujifilm, Japan). Protein levels were quantified using ImageJ software (http://rsbweb.nih.gov/ij/ ).
5
Protein Extraction and Western Blot Analysis
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Cells seeded in 6-well plates and treated with compounds were washed with phosphate-buffered saline (PBS) twice to remove media. Cells were then incubated in lysis buffer (Cell Signaling Technology) supplemented with 1 mM phenylmethylsulfonyl fluoride on ice for 30 min. Lysates were centrifuged at 14,000 × g for 10 min at 4°C, and the supernatants were collected. Protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Lysates were boiled with SDS sample buffer (Bio-Rad, Hercules, CA, USA) containing 100 mM dithiothreitol (DTT) for 5 min. Samples were subjected to SDS-polyacrylamide gel electrophoresis (Wako Pure Chemical Co., Osaka, Japan), transferred to a Polyvinylidene Difluoride (PVDF) membrane with an iBlot apparatus (Thermo Fisher Scientific), and immunoblotted with indicated antibodies. Protein-antibody complexes were detected using enhanced chemiluminescence detection reagents (PerkinElmer).
6
Cellular Fractionation and Western Blot
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Cells were seeded in 6-well tissue-culture treated plates for 48 h until 80% confluency prior to treatment. Whole cell extracts were prepared with Laemmli buffer (5% SDS, 10% glycerol, 0.5 M Tris-Cl) supplemented with protease and phosphatase inhibitors and equivalent amounts of proteins were separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) and blocked with 5% BSA in tris-buffered saline (TBS) supplemented with 0.1% Tween-20 for 1 h. Membranes were incubated with primary antibody overnight on a shaker at 4 ºC. They were then washed and incubated with secondary antibody for 1 h. Bands were visualized with the Enhanced Chemiluminescence Detection reagents (PerkinElmer).
Cells were lysed using the CERI and CERII reagents of the cytoplasmic-nuclear extraction kit (ThermoFisher), to obtain cytoplasmic fractions following treatment. Nuclear fractions were isolated by using 1× Laemmli buffer and sonication for a few seconds. Membrane was incubated overnight with active β-catenin (MilliporeSigma, Burlington, MA, USA), lamin A/C and tubulin antibodies.
Cells were lysed using the CERI and CERII reagents of the cytoplasmic-nuclear extraction kit (ThermoFisher), to obtain cytoplasmic fractions following treatment. Nuclear fractions were isolated by using 1× Laemmli buffer and sonication for a few seconds. Membrane was incubated overnight with active β-catenin (MilliporeSigma, Burlington, MA, USA), lamin A/C and tubulin antibodies.
7
Flagellin Quantification by Western Blot
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Flagellin levels were determined as described previously by SDS-PAGE and Coomassie brilliant blue staining [15 (link)]. Western blotting was performed to confirm the flagellin band. The flagellin samples on the SDS-PAGE gel were transferred to an Hybond-P membrane (GE Healthcare, Chicago, IL, USA). The blot was incubated with mouse polyclonal antiserum against FlaA, followed by sheep anti-mouse IgG conjugated with horseradish peroxidase (GE Healthcare, Chicago, IL, USA), and then developed using enhanced chemiluminescence detection reagents (PerkinElmer, Waltham, MA, USA).
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