Proteins were extracted with the CERAPHA kits and quantified as above. Twenty µg of protein from each sample was mixed 3:1 with 3x loading buffer (1M Tris-HCl pH 6.8, 20% SDS, 100% glycerol, β-mercaptoethanol and bromophenol blue) and boiled for 5min. Samples were loaded onto NuSep Tris-HEPES NH 12% precast gels and run at 110V for approximately 60min. Proteins were transferred onto a nitrocellulose membrane and probed using polyclonal antibody raised against selected wheat peptides showing strong distinctive antigenic capacity against TaAMY1, TaAMY2, and TaAMY3 (Genscript, USA). The antigen–antibody complexes were visualized by chemiluminescence (Sigma-Aldrich). The three polyclonal antibodies showed similar affinity for the same concentration of their respective peptides at the used dilution (1/2000).
Chemiluminescence
Manufactured by Merck Group
Sourced in United States, Germany, China, France, Japan
Chemiluminescence is a laboratory technique that involves the emission of light during a chemical reaction. It is a sensitive and versatile method used for quantitative and qualitative analysis in various scientific fields, including biochemistry, molecular biology, and analytical chemistry. The core function of chemiluminescence is to detect and measure the light produced as a result of a chemical reaction, providing valuable data and insights for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (39 mentions)
β actin, by Cell Signaling Technology (9 mentions)
Polyvinylidene fluoride membrane, by Merck Group (8 mentions)
Bca assay, by Thermo Fisher Scientific (7 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (6 mentions)
Ripa buffer, by Beyotime (6 mentions)
N cadherin, by Cell Signaling Technology (5 mentions)
E cadherin, by Cell Signaling Technology (5 mentions)
Protease inhibitor cocktail, by Roche (5 mentions)
Bca protein assay kit, by Beyotime (5 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Ab125066, by Abcam (4 mentions)
Imaging software, by GE Healthcare (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Protease inhibitor, by Roche (4 mentions)
Ripa lysis buffer, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by GE Healthcare (4 mentions)
β actin, by Merck Group (3 mentions)
172 protocols using chemiluminescence
1
Wheat Amylase Protein Detection
2
Protein Extraction and Western Blot
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Overnight cultures were diluted to OD600 of ~0.02 in media with 0.2% arabinose with 75 µg/mL gentamicin. Culture density was monitored by OD600. At OD600 ~1.5, cells were collected by centrifugation and resuspended in 1/15 vol of 1x PBS. Proteins were immediately precipitated by a modified MeOH/CHCl3 procedure (sample/MeOH/CHCl3: 1/1/0.25 [Wessel and Flügge, 1984 (link)]). Samples were separated on 12% SDS-PAGE, transferred to PVDF membranes and blocked with Tris buffered saline (50 mM Tris, pH7.4, 200 mM NaCl) with 5% non-fat milk. HA-tags were detected using mouse anti-HA (Sigma H9658) and goat anti-mouse IgG-HRP conjugate (Sigma A9917). HRP signal was developed using chemiluminescence per manufacture protocol (Sigma).
3
Measurement of Intracellular and Extracellular ROS
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The intracellular production of O2− in EBV-B cells was assessed by chemiluminescence (Sigma-Aldrich; van de Geer et al., 2018 (link)). We stimulated 5 × 105 cells with PMA (400 ng/ml) or heat-killed S. aureus cells (Pansorbin, 2 mg/ml; Sigma-Aldrich) and light production was monitored every 5 min with a Victor X4 plate reader. NADPH oxidase activity was assessed in EBV-B cells, MDMs, and MDDCs by measuring extracellular H2O2 release with an Amplex Red Kit (Thermo Fisher Scientific), as previously described (van de Geer et al., 2018 (link); Bustamante et al., 2011 (link); Conti et al., 2015 (link)). In brief, 3 × 104 cells were stimulated with PMA (400 ng/ml), and H2O2 release was quantified with a Victor X4 plate reader (PerkinElmer). MDMs or MDDCs were cultured for 16 to 18 h before the experiment, in the presence of 5 × 103 IU/ml IFN-γ (Imukin, Boehringer Ingelheim) or 1 µg/ml LPS (Salmonella minnesota; Sigma-Aldrich), respectively. ROS production by neutrophils and monocytes was quantified with the Burstest (Phagoburst kit; BD) containing PMA and opsonized E. coli bacteria. The assay was performed according to the manufacturer’s instructions, and the amount of rhodamine 123 (mean fluorescence intensity) was assessed by flow cytometry with a Gallios FACS Analyzer. DHR oxidation is expressed as a percentage of the rhodamine 123 produced by the corresponding travel controls.
4
VEGFR2 Protein Expression Analysis
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Cells were lysed, total protein concentration was measured by bicinchoninic acid method. Thirty micrograms of protein was electrophoresed on 8% SDS–PAGE gel and transferred to a PVDF (polyvinylidene fluoride) membrane (Millipore, Billerica, MA, USA). After blocking the membrane, it was incubated with primary antibody (rabbit anti-VEGFR2 and anti-phospho-VEGFR2 monoclonal antibodies, 1:1000; catalog no.8696 S, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. The membrane was washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibody. The protein bands were detected by chemiluminescence (Sigma) and visualized using BioImaging Systems. Protein expression was normalized to β-actin levels.
5
Protein Profiling of Rat Ovaries
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Total protein isolated from rat ovaries was used for Western blotting. Extracts containing equivalent quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 7.5-12% gels) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The antibodies used included β-actin (Sigma-Aldrich, USA); PI3K, Akt, mTOR, PCNA, cleaved-caspase3, Bax, Bcl2, and phospho-CaMKII (Abcam, Britain); phospho-Akt, GDF9, BMP15, and BMPR2 (Abclonal, China); Smad3 (Beyotime, China); caspase3 (Bioss, China) and CaMKII (Cell Signaling Technology, USA). The protein band corresponding to protein specifically bound by the primary antibody was detected by an anti-rabbit or an anti-mouse IgG-HRP-linked antibody (Sigma-Aldrich, USA). Antibody-reactive bands were visualized using chemiluminescence (Sigma-Aldrich, USA).
6
Western Blot Analysis of Cellular Proteins
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Protein samples were isolated from ASMCs cells using RIPA lysis buffer (Beyotime, Beijing, China). Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes. The membranes were blocked with skimmed milk and probed with specific antibodies: anti-Bax (ab216494) and anti-Bcl-2 (ab59348) (1:2000; Abcam, Cambridge, UK); anti-cleaved caspase-3 (ab2302) and anti-cleaved caspase-9 (ab2324) (1:2500, Abcam); anti-fibronectin (ab2413) and anti-collagen I (ab34710) (1:3000, Abcam); anti-MMP9 (ab38898) and anti-versican (ab19345) (1:3500, Abcam); anti-extracellular receptor kinase (ERK) (ab17942) and anti-phosphorylated (p)-ERK (ab278538) (1:4000, Abcam); and anti-Jun N-terminal kinase (JNK) (ab179461), anti-p-JNK (ab4821), anti-p38 (ab170099), antip-p38 (ab4822), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab9485) (1:4500, Abcam). Following incubation with horseradish peroxidase-conjugated secondary antibody (ab205719; ab205718) (1:5000, Abcam) and tetramethylbenzidine, the protein bands were visualized using chemiluminescence (Sigma-Aldrich). The Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used to quantify the protein bands with GAPDH as a reference.
7
Western Blot Analysis of AMPK, ACC, pACC, and PKCα
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AMPK, ACC, pACC and PKCα were determined by immunoblotting using 10% SDS-PAGE.
Membranes were blocked with 5% milk in 1X PBST and incubated overnight at 4°C with anti-AMPK (Abcam), anti-ACC or anti-pACC (Abcam)and anti-PKC ߙ (Santa Cruz) antibodies.
Membranes were then incubated with the HRP-conjugated anti-rabbit IgG and visualized by chemiluminescence (Sigma-Aldrich).
Membranes were blocked with 5% milk in 1X PBST and incubated overnight at 4°C with anti-AMPK (Abcam), anti-ACC or anti-pACC (Abcam)and anti-PKC ߙ (Santa Cruz) antibodies.
Membranes were then incubated with the HRP-conjugated anti-rabbit IgG and visualized by chemiluminescence (Sigma-Aldrich).
8
Immunoblotting Analysis of Inflammasome Activation
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Proteins isolated from HK-2 cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membrane. The membrane was blocked and probed with specific antibodies: anti-NLR family pyrin domain containing 3 (NLRP3) and anti-N-terminal domain of gasdermin D (GSDMD-N) (1:2,000; Abcam, Cambridge, UK); anti-caspase-1 and anti-IL-1β (1:2,500; Abcam); anti-IL-18 and anti-thioredoxin-interacting protein (TXNIP, 1:3,000; Abcam); and anti-5' adenosine monophosphate-activated protein kinase (anti-AMPK), anti-phosphorylated AMPK (p-AMPK) and anti-β-actin (1:3,500; Abcam). Following incubation with horseradish peroxidase-conjugated secondary antibody (1:4,000; Abcam) and tetramethylbenzidine, the protein bands were visualized using chemiluminescence (Sigma-Aldrich).
9
Investigating NF-κB Signaling in RA FLSs
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For each Western blot analysis, 2×105 cells were seeded. When subconfluence reached 80%, RA FLSs were pretreated with DMSO (5%) or NH125 for 24 h or transfected with eEF2K siRNA or si-C. Then, RA FLSs were stimulated with or without TNF-α (10 ng/mL) for 15 min. Proteins in RA FLSs were separated by SDS-PAGE (12%) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were incubated for 12 h at 4° C with the following primary antibodies: phosphorylated-IKK, IKK, IκBα, phosphorylated-IκBα, phosphorylated-AKT, AKT and β-actin (1:1000 dilution, all from Cell Signaling Technology, Danvers, MA, USA). Then, the PVDF membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 5000 dilution, Cell Signaling Technology) at room temperature for 1 h. The protein bands were visualized with chemiluminescence (Millipore, Boston, MA, USA). The membrane was scanned and densitometry quantified by ImageJ analysis system (National Institutes of Health). The intensity of each protein was First normalized to β-actin bands and represented as a ratio of controls.
10
Isolation and Analysis of Peritoneal Macrophages
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The peritoneal macrophages were cultured in DMEM medium with 10% FCS in 12-well plates. Cells were treated with IL-4 and the indicated reagents for the indicated time depending on the experiment purpose. After stimulation, the cells were washed once in cold PBS, lysed in RIPA buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA pH7.4) with protease and phosphatase inhibitor cocktails (Sigma) for 10 min on a rocker at 4°C. Cells were scraped, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatants were mixed with 5× protein-loading buffer (26 (link)). Protein concentration is determined using a BCA assay. Proteins samples were analyzed on SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore). PVDF membrane was blocked with TBST (100 mM Tris–HCl pH7.5, 150 mM NaCl, 0.05% Tween20) with 5% non-fat dried milk for 1 h and then incubated with primary antibodies overnight on a shaker at 4°C. The appropriate HRP-coupled secondary antibody was used and detected through chemiluminescence (Millipore) (27 (link)). β-Actin was used as a protein-loading control. PVDF membranes were stripped, washed in TBST, and immunostained with the other antibody.
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