Two batches of bacterial samples were removed from storage at −80 °C and lysed immediately using B-PER™ Bacterial Protein Extraction Reagent (ThermoFisher Scientific; Waltham, MA, USA) containing 1 mM PMSF, 1 mM EDTA, and 120 μg/mL DNaseI. The suspensions were inverted at room temperature for 10 min, and the cell debris was removed by centrifuging at 13,000× g for 10 min. Two micrograms of cell lysates were separated by 12.5% SDS-PAGE, transferred onto a PVDF membrane, and blocked with 5% skim milk at 4 °C overnight. The blots were then hybridized with chicken anti-PirAvp or chicken anti-PirBvp polyclonal antibodies (1:5000 diluted with 5% skim milk). After 1 hour of incubation at room temperature, the blots were washed 3 times with PBST solution (1× PBS contained 0.1% Tween-20). The blots were further incubated with donkey anti-chicken-HRP conjugated secondary antibody (Jackson; West Grove, PA; 1:10,000 diluted with 5% skim milk) at room temperature for 1 h. Following 3 more washes, the protein bands were visualized using a chemiluminescence reagent (GE Healthcare; Chicago, IL, USA) and detected with an Amersham Imager 600 (GE Healthcare; Chicago, IL, USA). For loading controls, 8 μg of cell lysates were separated with another 12.5% SDS-PAGE, and stained with Coomassie blue.
Chemiluminescence reagent
Manufactured by GE Healthcare
Sourced in United States
Chemiluminescence reagent is a laboratory product used to facilitate chemiluminescence-based analytical techniques. It is designed to produce light through a chemical reaction, which can be detected and measured to quantify the presence or concentration of specific analytes in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit, by Bio-Rad (7 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Amersham imager, by Cytiva (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Amersham imager 600, by GE Healthcare (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Complete protease inhibitor tablet, by Roche (2 mentions)
Quantity one v4, by Bio-Rad (2 mentions)
Polyvinylidene difluoride, by Merck Group (2 mentions)
Horseradish peroxidase, by GE Healthcare (2 mentions)
B per bacterial protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Bca protein kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Cell lysis buffer, by Merck Group (1 mentions)
Versadoc 5000 system, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Immobilon membrane, by Merck Group (1 mentions)
39 protocols using chemiluminescence reagent
1
Immunoblotting of PirA and PirB Proteins
2
Protein Expression Analysis by Western Blot
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The protein concentration in cell lysates was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Lysates were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore Corporation). Membranes were blocked with 5% non-fat milk and incubated with specific primary antibodies at 4°C overnight. This was followed by incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for hybridization. Protein bands were visualized with a chemiluminescence reagent (GE Healthcare Biosciences).
3
Protein Quantification and Western Blot Analysis
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To determine protein concentration, a protein assay kit (Bio-Rad Laboratories, Hercules, CA) was used. After subjection to SDS-PAGE, the samples were transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore Corporation). Then the membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with specific primary antibodies overnight at 4°C. Finally, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized with a chemiluminescence reagent (GE Healthcare Biosciences).
4
Protein Extraction and Analysis Protocol
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Cells were lysed by lysis buffer (150 mmol/L NaCl, 1% NP-40, 50 mM Tris–HCl with 1 mM PMSF and protease inhibitor, and dephosphorylate inhibitor tablets mixture) for total protein collection. As per the procedure of the Nuclear-Cytosol Extraction Kit (Key GEN Technologies Inc., Jiangsu, China) to separate the nuclear and cytoplasmic proteins from total cellular proteins. The protein concentrations of the extracts of each group were quantified using the BCA protein kit (Thermo, USA). A same amount of protein was fractionated via SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). Membranes were blocked using 5% non-fat dry milk in 1× TBST for 1 h and then incubated with primary antibodies overnight at 4 °C. Then, the membrane was washed with 1× TBST and incubated with the corresponding secondary antibody. Protein bands were visualized using the chemiluminescence reagent (GE Healthcare Life Science) with Da Vinci software.
5
Western Blot Analysis of ESCC Cells
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Protein concentrations of ShODC or DFMO-treated ESCC cell lysates were determined using a protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA). Total proteins (20 to 100 μg) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). After blocking in 5% non-fat milk, the membranes were probed with specific primary antibodies overnight at 4 °C, then washed 3 times with TBS-Tween 20 followed by incubation at room temperature 1 h with a horseradish peroxidase (HRP)-conjugated secondary antibody. The protein bands were visualized with a chemiluminescence reagent (GE Healthcare Biosciences, Pittsburgh, PA).
6
Protein Quantification and Western Blot
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Sample protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA). Total proteins (20 to 100 μg) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). After blocking in 5% milk, the membranes were probed with specific primary antibodies overnight at 4°C, washed 3 times with TBS-Tween 20, and then incubated with a horseradish peroxidase (HRP) conjugated secondary antibody at room temperature 1 h for hybridization. The protein bands were visualized with a chemiluminescence reagent (GE Healthcare Biosciences, Pittsburgh, PA).
7
Western Blot Protein Detection
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Target proteins were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The cells were lysed using cell lysis buffer (Sigma-C2978) with protease inhibitors (Complete Protease Inhibitor Tablets; Boehringer Mannheim, Indianapolis, IN, USA). The proteins were separated using 10% SDS-PAGE and electrotransferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight at 4 °C with a primary antibody against a specific target and chemiluminescent detection was uses a horseradish peroxidase-conjugated secondary antibody (1:5000). The signals were visualized with a chemiluminescence reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and detected using the VersaDoc 5000 system (Bio-Rad Laboratories, Hercules, CA, USA) [33 (link)].
8
Western Blot Analysis of Lung Proteins
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The total proteins of lung or cells were extracted using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, China) and the protein concentrations were measured with BCA protein assay kit (Beyotime Institute of Biotechnology, China). Equal amounts of total protein were loaded onto 10% SDS PAGE gels at 100 V for 60 minutes and electro transferred to PVDF membranes. After blocked in 5%no‐fat milk at room temperature for 2 hours, the membranes were incubated with the diluted antibodies at 4°C overnight. After washing with TBST, the membranes were probed with horseradish peroxidase‐conjugated secondary antibody at room temperature for 1 hour. Then proteins were detected by chemiluminescence reagent (GE Healthcare). Expression levels of the proteins were normalized by GAPDH or β‐actin as a loading control. Protein band density was quantified using Bio‐Rad Quantity One v4.62. All experiments were conducted in triplicates and repeated three times. All data and images were analysed blindly.
9
Cell Lysis and Immunoblotting Protocol
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Following the chemical treatments, cells were washed with PBS and lysed with RIPA buffer containing 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% SDS, 1 mM DTT, phosphatase inhibitor mix (Nacalai Tesque) and protease inhibitor cocktail (Nacalai Tesque) on ice. The cell lysates were subjected to immunoblotting with the indicated antibodies, and the immune complexes were detected using a chemiluminescence reagent (GE Healthcare).
10
Protein Expression Analysis in Colon Tissues
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Colon tissues from AC and AMC mice or CRC cell lines were lysed in RIPA lysis buffer along with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Equal amounts of total protein were loaded and separated by 10-12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Due to its high molecular weight, MUC4, MUC5AC and MUC1 were resolved in 2% SDS-agarose gel. After blotting, membranes were blocked for 1 hr in 5% non-fat dry milk containing phosphate-buffered saline and 0.1% Tween 20 (v/v) and incubated with indicated primary antibodies overnight at 4° C, followed by incubation for 1 hr with respective secondary antibodies and protein bands were detected with a chemiluminescence reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).
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