Human bronchial epithelial cells were lysed in lysis buffer and subjected to SDS-PAGE and a Western blot analysis38 (link). Blots were reacted for 2 h with monoclonal antibody diluted at 1:1000 and with the respective HRP-conjugated secondary antibodies diluted at 1:2000. After each antibody reaction, membranes were washed 3 times with 0.1% TBS-Tween, and blots were visualized with Super Signal West Pico chemiluminescence substrate (Thermo Fisher Scientific, Inc.). Images were captured by LAS-4000 (GE HealthCare Dharmacon, Inc.). The band intensity was quantified using the Multi Gauge software program (FUJIFILM, Inc., Tokyo, Japan). For the detection of MUC5AC protein secretion into the medium, slot blotting was performed as previously described39 (link).
Multi gauge software program
Manufactured by Fujifilm
Sourced in United States, Japan
The Multi-Gauge software program is a data analysis tool designed to work with Fujifilm's line of surface roughness measurement instruments. The core function of the software is to provide users with the ability to analyze and process the data collected from these measurement devices.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (7 mentions)
β actin, by Merck Group (5 mentions)
Lysis buffer, by Kurabo (3 mentions)
Supersignal west pico chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Matrix metalloproteinase 2 mmp 2, by Bioss Antibodies (1 mentions)
Mmp 9, by Abnova (1 mentions)
A5441 100ul, by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
F4 80, by Abcam (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
9 protocols using multi gauge software program
1
Western Blot Analysis of MUC5AC Secretion
2
Western Blot Analysis of Colon26 Cell Proteins
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Cultured Colon26 cells (10
6cells) were homogenized with a lysis buffer containing 0.5% Nonidet P-40, 10% glycerol, 137 mM NaCl, 2 mM ethylenediaminetetraacetic acid, and 50 mM Tris-HCl buffer (pH 8.0). After centrifugation at 8,000 ×
gfor 10 minutes, the supernatant fraction was collected, and the total protein concentration of the cell extract was measured using Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, California, United States) and stored at −80°C until analysis. Samples (0.25–2.5 μg total protein/lane) were subjected to 10% polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. The electrophoresed protein was transferred to an Immobilon membrane (Life Technologies, Carlsbad, California, United States), subsequently blocked with 5% skim milk at 4°C overnight, and incubated with anti-mouse TF monoclonal antibody (1:1,000; Bioss Antibodies Inc., Woburn, Massachusetts, United States) at 25°C for 1 hour. The membrane was then treated with a horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, Maryland, United States). Immune complexes were detected using ImmunoStar Zeta regent (Wako, Osaka, Japan), and images were acquired using the Multi-Gauge software program (Fujifilm, Greenwood, South Carolina, United States).
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Cultured Colon26 cells (10
6cells) were homogenized with a lysis buffer containing 0.5% Nonidet P-40, 10% glycerol, 137 mM NaCl, 2 mM ethylenediaminetetraacetic acid, and 50 mM Tris-HCl buffer (pH 8.0). After centrifugation at 8,000 ×
gfor 10 minutes, the supernatant fraction was collected, and the total protein concentration of the cell extract was measured using Bradford Protein Assay Kit (Bio-Rad Laboratories, Hercules, California, United States) and stored at −80°C until analysis. Samples (0.25–2.5 μg total protein/lane) were subjected to 10% polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. The electrophoresed protein was transferred to an Immobilon membrane (Life Technologies, Carlsbad, California, United States), subsequently blocked with 5% skim milk at 4°C overnight, and incubated with anti-mouse TF monoclonal antibody (1:1,000; Bioss Antibodies Inc., Woburn, Massachusetts, United States) at 25°C for 1 hour. The membrane was then treated with a horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, Maryland, United States). Immune complexes were detected using ImmunoStar Zeta regent (Wako, Osaka, Japan), and images were acquired using the Multi-Gauge software program (Fujifilm, Greenwood, South Carolina, United States).
3
Western Blot Analysis of Retinal Markers
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We performed Western blot analysis as previously described [17 (link)]. Briefly, the supernatant samples of the retina (equal amounts of protein samples) were loaded onto a BIS-TRIS Blot Gel (Life Technologies, Carlsbad, CA, USA) and electrophoresed. Following separation, the proteins were transferred to a nitrocellulose membrane and incubated with 5% skimmed milk at 4°C overnight. After blocking, the membranes were incubated at 25°C for 1 h with primary antibodies against iNOS (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), matrix metalloproteinase-2 (MMP-2) (1:1,000; Bioss Inc., Woburn, MA, USA), MMP-9 (1:1,000; Abnova Co., Taipei, Taiwan), vascular endothelial growth factor (VEGF) (1:1,000; Abnova Co.), macrophages (F4/80, 1:1,000; Bio-Rad, Raleigh, NC, USA), β-amyloid (1:1,000; Rockland Immunochemicals Inc., Gilbertsville, PA, USA), and β-actin (1:5,000; Sigma-Aldrich, St. Louis, MO, USA). Bands on the membranes were visualized using a horseradish peroxidase-conjugated secondary antibody (Life Technologies, Frederick, MD, USA) and ImmunoStar Zeta reagent (Wako, Osaka, Japan). Images were acquired using the Multi-Gauge Software program (Fujifilm, Greenwood, SC, USA).
4
Western Blotting Analysis of Fibroblast Markers
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Western blotting was performed as previously described.14 (link) Briefly, the samples were separated by electrophoresis and the membranes were incubated at 25°C for 1 h with primary antibodies against S100A4, a fibroblast-specific marker (1:1000; RB-1804-A0, Thermo Fisher Scientific, Waltham, MA, USA), collagen type I (1:1000; 234,167, Millipore, Billerica, MA, USA), cleaved caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-9 (1:1000; Cell Signaling Technology), or β-actin as the loading control (1:5000; A5441-100UL, Sigma-Aldrich Corp., St. Louis, MO, USA). The membranes were then treated with a horseradish peroxidase-conjugated secondary antibody (1:1000; Novex, Frederick, MD, USA). Images of the membranes were acquired with the Multi-Gauge Software Program (Fujifilm, Greenwood, SC, USA).
5
Protein Expression Analysis in Liver Samples
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The collected liver samples were homogenized with a lysis buffer and centrifuged at 1000 rpm at 4 °C for 20 min to collect the supernatant. SDS-PAGE was performed as previously described [14 (link)]. Protein was subsequently blocked for 1 h with 5% skim milk at room temperature. The membranes were washed with Tris-buffered saline–0.1% Tween 20 three times and were incubated at room temperature for 1 h with primary antibodies against Iba1 (marker of macrophage; 1:1000; Wako), chemokine receptor 7 (CCR7) (marker of M1 macrophage; 1:1000; Abcam, Cambridge, MA, USA), and β-actin as a loading control (1:5000; Sigma-Aldrich, St. Louis, MO, USA). Next, the membranes were washed and treated with horseradish peroxidase-conjugated secondary antibody (1:1000; Novex, Frederick, MD, USA), and the immune complexes were detected using ImmunoStar Zeta regent (Wako). The images were acquired using the Multi-Gauge software program (Fujifilm, Greenwood, SC, USA).
6
Western Blot Analysis of Brain Proteins
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The whole-brain samples were homogenized in lysis buffer (Kurabo, Osaka, Japan) and centrifuged at 8000 x g for 10 min. The supernatant from each sample was separated on SDS-PAGE and western blot was performed as described previously 17 (link). Briefly, 10 μg of protein was separated by electrophoresis and transferred to nitrocellulose membranes. Subsequently, the membranes were incubated at 25°C for 1 h with the following primary antibodies; against CRHR-1 (1:1000; GeneTex Inc., Irvine, CA, USA), CRHR-2 (1:1000; Novus Biologicals, Littleton, CO, USA), β-amyloid (1:1000; Rackland Inc., Gilbertsville, PA, USA), Iba (marker of microglia, 1:1000, Wako, Osaka, Japan), IL-6 (1:1000; Abcam, Cambridge, UK), F4/80 (marker of macrophages, 1:1000, Abcam) or β-actin as the loading control (1:5000; Sigma-Aldrich, St. Louis, MO, USA). Next, the membranes were treated with horseradish peroxidase-conjugated secondary antibody (1:1000; Novex, Frederick, MD, USA), and the immune complexes were detected using ImmunoStar Zeta regent (Wako). The images were acquired using the Multi-Gauge software program (Fujifilm, Greenwood, SC, USA).
7
Western Blot Protein Analysis Protocol
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Cells were washed with PBS and lysed in buffer (50 mM Hepes, 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 50 mM NaF, 10 mM NaPPi, 2 mM NaVO3, 10 mM EDTA, 2 mM EGTA, 1 mM PMSF, and 10 μg/ml leupeptin) on ice for 10 min. Protein lysate from the cells was mixed with loading buffer, denatured for 10 min at 65°C, separated on SDS-PAGE, and later transferred to a PVDF membrane. After blocking (5% dry milk in Tris-buffered saline-T), membranes were incubated at 4°C overnight with primary antibodies. After incubation with secondary antibodies, the signals were visualized by chemiluminescence using a chemiluminescence (ECL) detection device (Millipore, Billerica, MA, USA). Optical densities were visualized and measured as arbitrary units via an LAS3000 Imager (Fugifilm, Greenwood, SC, USA). The results were normalized to GAPDH using the Multi-gauge software program (Version 22.0, Fujifilm, Greenwood, SC, USA).
8
Western Blot Analysis of RORα in Skin
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The skin samples were homogenized in a lysis buffer (Kurabo, Osaka, Japan) and centrifuged at 8,000 g for 10 min. We performed a Western blotting analysis as previously described [23 (link)]. Briefly, the membranes were incubated at 25oC for 1 h with primary antibodies against RORα (1:1,000; GeneTex, Irvine, CA, USA) or β-actin (1:5,000; Sigma-Aldrich, St. Louis, MO, USA). The membranes were then treated with a horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). The immune complexes were detected using an ImmunoStar Zeta reagent (Wako, Osaka, Japan), and images were acquired using the Multi-Gauge software program (Fujifilm, Greenwood, SC, USA).
9
Western Blot Analysis of Tissue Proteins
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The organ samples were homogenized in lysis buffer (Kurabo, Osaka, Japan) and centrifuged at 8000× g for 10 min. Western blot analysis was performed as described previously [48 (link)]. The membranes were incubated at room temperature for 1 h with primary antibodies against TF (1:1000; Bioss, Woburn, MA, USA), syndecan-4 (1:1000; LifeSpan BioSciences, Seattle, WA, USA), IL-6 (1:1000; R&D Systems, Minneapolis, MN, USA), or β-actin as a loading control (1:5000; Sigma-Aldrich, St. Louis, MO, USA). Thereafter, the membranes were washed and incubated with a horseradish-peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). The immune complexes were detected using ImmunoStar Zata reagent (Wako, Osaka, Japan), and images were acquired using the Multi Gauge software program (Fujifilm, Greenwood, SC, USA).
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