(Conditioned mediums) CMs were collected from C4‐2 culture only, HH culture only, and C4‐2‐HH coculture for 48 hours. Briefly, 1 mL of these supernatants was incubated with the RayBio1 Human Cytokine Antibody Array 3 (Catalog no. AAH‐CYT‐3) (RayBiotech, Norcross, GA). Arrays were processed according to the manufacturer's protocols and evaluated using the chemiluminescence system (Thermo Fisher Scientific).
Chemiluminescence system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Chemiluminescence system is a laboratory equipment designed to detect and quantify light-emitting chemical reactions. It serves as a tool for researchers and scientists to analyze various samples and biological processes that generate luminescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Protease inhibitor, by Merck Group (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Immobilon p, by Merck Group (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Hrp conjugated secondary antibody, by LGC (2 mentions)
Tgx gel, by Bio-Rad (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Ab205718, by Abcam (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Gel dock system, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Bicinchoninic acid kit, by Bio-Rad (1 mentions)
42 protocols using chemiluminescence system
1
Cytokine Profiling of Conditioned Mediums
2
Isolation and Analysis of FLAG-tagged Proteins
3
Protein Detection via Western Blot
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Proteins were separated by SDS–polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane (Immobilon-P, EMD Millipore) by liquid transfer, and the Western blots were probed using the appropriate primary antibodies (1:1000) followed by alkaline phosphatase secondary antibodies (1:5000). The signals were detected using a chemiluminescence system (Thermo Fisher Scientific), followed by Gel Dock system (Bio-Rad) imaging.
4
Western Blot Analysis of Signaling Proteins
5
Sertoli Cell Protein Extraction and Western Blot
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Sertoli cell washed with ice-cold PBS, and lysed in extraction buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.4) supplemented with protease and phosphatase inhibitor mixtures and PMSF (Sigma, MO), sonicating, and centrifuging to obtain the clear supernatant. Lysates were stored at -80°C until used. For immunoblot analysis, lysates (30 μg of protein) were separated by SDS-polyacrylamide gel, blotted onto PVDF membrane (Immobilon-P; Millipore), and blocked in TBS/0.1% Tween-20/5% BSA (TBS, 25 mM Tris, 150 mM NaCl, pH 7.4) for 1 h, followed by primary antibodies incubation for overnight at 4°C. After washing in TBS/0.1% Tween-20, membranes were subjected to incubation with corresponding HRP-conjugated secondary antibodies. The immunoblot bands were visualized with ECL kit and read using chemiluminescence system (Thermo Scientific, Waltham, MA). Antibodies and dilutions were listed in Table 1 .
6
Western Blot and Immunoprecipitation Protocols
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The colon tissues or Caco-2 cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) containing 1% protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein concentration of each sample was determined by the Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China), and equal protein was separated on 8%–10% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked by 5% skim milk powder in Tris-buffered saline-Tween 20 (TBST; 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 [pH 7.6]) for 1 h and incubated with primary antibodies at 4°C overnight. After washing and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, the blots were visualized by the chemiluminescence system (Thermo Fisher Scientific) and quantified by ImageJ software. For immunoprecipitation, nonspecific binding was precleared with A/G agarose beads (Santa Cruz Biotechnology, CA, USA) and then the lysates were coincubated with indicated primary antibodies and A/G agarose beads at 4°C for 4 h. The beads containing the bound proteins were washed with PBS three times and boiled in lysis buffer at 95°C for 10 min. The supernatant was subjected for western blotting analysis.
7
Western Blot Protein Analysis Protocol
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Cells were lysed with an appropriate volume of radioimmunoprecipitation buffer supplemented with protease and phosphatase inhibitor cocktails, and the protein concentrations were determined by bicinchoninic acid assays with BSA (all CWBIO; CW2200S, CW2383, CW0017) as the standard. A total of 30 ng/20 µl protein was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA; C3117). Following blocking with Tris-buffered saline containing 5% BSA diluted in TBS with Tween-20 for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies diluted according to the instructions, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immunoreactive bands were detected using a chemiluminescence system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and quantified using ImageJ 1.43 (National Instituted of Health, Bethesda, MD, USA).
8
Western Blot Protein Quantification
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HBVSMCs or cerebrovascular tissues were lysed using RIPA buffer (Beyotime) containing 1% protease and phosphatase inhibitors (Merck). Nuclear proteins were extracted using a Nuclear/Cytosol Fractionation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The protein concentration of each sample was determined using a bicinchoninic acid kit (BioRad). Equal amounts of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 8–10% gels and then transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% non-fat milk powder in TBST consisting of 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 (pH 7.6) and incubated with the appropriate antibodies at 4 °C overnight. Afterwards, the blots were visualized with HRP-conjugated secondary antibodies followed detection with a chemiluminescence system (Thermo Fisher Scientific). Image quantification was performed using ImageJ software.
9
Western Blot Analysis of KNL1 and GAPDH
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Cells and tissues were lysed using RIPA buffer (Thermo Fisher Scientific, Inc.). Lysates were electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific, Inc.) and transferred onto polyvinylidene difluoride membranes (Thermo Fisher Scientific, Inc.). After blocking for 1 hour in 5% skimmed milk, the membranes were incubated overnight at 4°C with rabbit antibodies against KNL1 (dilution, 1:500; Biorbyt Ltd.) and GAPDH (dilution, 1:800; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), washed four times in TBST, and then incubated at room temperature for 1 hour with a horseradish peroxidase-conjugated secondary antibody (dilution, 1:10,000; catalog no., 323-065-021; Jackson ImmunoResearch, Inc., West Grove, PA, USA). Protein bands were visualized using a chemiluminescence system (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. The band intensities were quantified using Image-Quant Software v3.0 (LI-COR Biosciences, Lincoln, NE, USA). This experiment was repeated in triplicate.
10
Western Blot Protein Analysis
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The protein sample was mixed with loading buffer and heated at 95°C for 5 min. 20 μg samples and standard protein (Bio-Rad, Hercules, CA, USA) were loaded on a gradient polyacrylamide gel (4–10%, Genscript, Piscataway, USA). The gel was run at 110 V for 120 min (4°C) and then the proteins were transferred onto a polyvinylidene difluoride membrane. After transfer at 120 V for 90 min, the membrane was blocked in 5% bovine serum albumin for 60 min at room temperature. Subsequently, the membrane was incubated with the primary antibodies overnight at 4°C and then incubated with anti-rabbit IgG for 60 min. The detection was preformed using a chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA), and the bands were analyzed using the Quantity One system (Version 4.6.2).
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