Protein was collected from 2 × 106 cells treated with IL-9 for 48 h using a lysis buffer containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were centrifuged at 14,000 rpm at 4°C for 15 min. Protein samples were mixed with 5x sample buffer (4 : 1 ratio) and heated at 95°C for 10 min and then separated on 10% sodium dodecyl sulfate-polyacrylamide gels. Following electrophoresis for 90 min, the proteins were transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA). The membranes were blocked for 1 h at room temperature with 5% nonfat milk in 0.1% TBS-Tween 20 and then incubated with anti-β-catenin (1 : 2000 dilution; Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with LI-COR IRDye 680-labeled secondary antibodies (Rockland Immunochemicals, Gilbertsville, PA, USA) for 1 h at room temperature. Signals were detected with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and quantitated using the FluorChem 8900 system (Alpha Innotech, San Leandro, CA, USA).
Fluorchem 8900 system
Manufactured by Bio-Techne
Sourced in United States
The Fluorchem 8900 system is a compact and versatile fluorescence imaging platform designed for a wide range of applications in life science research. It offers high-sensitivity detection and supports a variety of fluorescent dyes and probes, enabling researchers to visualize and analyze diverse biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (2 mentions)
Anti β catenin, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Complete protease inhibitor mixture, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Peroxidase conjugated secondary antibody, by Boster Bio (1 mentions)
Phosphorylated ikk, by Cell Signaling Technology (1 mentions)
Phosphorylated iκbα, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Rabbit anti stat3, by Abcam (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Ecl chemiluminescent detection system, by Thermo Fisher Scientific (1 mentions)
Sc 69879, by Santa Cruz Biotechnology (1 mentions)
Ab5864, by Merck Group (1 mentions)
5 protocols using fluorchem 8900 system
1
Quantitative Analysis of β-Catenin Protein
2
Quantification of Myelin Basic Protein Isoforms
3
Western Blot Protocol for Protein Analysis
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The method of Western blotting analyses was performed as described previously.17 (link) Bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA) was used to detect protein concentrations. Equal amounts of protein were solubilized in Laemmli buffer (pH 6.8, 62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.0625‰bromophenol blue), boiled for 5 minutes, separated by 12% SDS-PAGE, and transferred to nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were probed with the following primary antibodies: phosphorylated-IKK, IκBα, phosphorylated-IκBα and β-actin (all from Cell Signaling Technology, Danvers, MA, USA) in TBS/Tween-20 containing 5% non-fat milk at 4°C overnight. The membranes were incubated with the secondary antibodies for 1 hr at room temperature. A peroxidase-conjugated secondary antibody (1:5000 dilution, BOSTER Biological Technology Co. Ltd, Wuhan, China) and enhanced chemiluminescence Western blot detection reagents (NCI4106, Pierce, ThermoFisher) were used to visualize the target proteins. Each blot is representative of at least 3 independent experiments. The film was scanned by a high-resolution scanner, densitometry was performed using AlphaEaseFc software on a Fluorchem 8900 system (Alpha Innotech, San Leandro, CA, USA).
4
Quantification of STAT3 and β-catenin
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After treatment with IL-22 for 48 h, the protein was extracted from cell lysates in the lysis buffer containing protease inhibitors and phosphatase inhibitors (Sigma, USA). The concentration of cellular protein was determined by the Pierce BCA assay (Thermo Fisher Scientific, Rockford, USA). Total protein extracts were separated by electrophoresis for 90 min and transferred onto a polyvinylidene fluoride membrane (Merck Millipore, USA). The primary antibodies used were rabbit anti-STAT3 (1:3000, Abcam, UK), anti-p-STAT3 primary antibody (1:2000, Abcam) and anti-β-catenin (1:2000, Abcam). LI-COR IRDye 680-labeled secondary antibody was used (Rockland Immunochemical, Gilbertsville, PA). The signals were detected and quantified by using Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE) and Fluorchem 8900 system (Alpha Innotech, San Leandro, CA).
5
Quantification of Demyelinated Protein Levels
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Detailed methods are described in our previous studies (Zhan et al., 2008 (link), 2014 (link), 2015 (link)). Briefly, frozen tissues were homogenized in ice-cold RIPA buffer containing a complete protease inhibitor (Sigma). Homogenates were centrifuged at 14,000 × g for 30 min at 4°C. Protein (12.5 μg each) from the supernatant was loaded on 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to the nitrocellulose membrane. The primary antibody included rabbit polyclonal against dMBP (AB5864, 1:1,000 dilutions; Millipore). NIH Image J software was used to quantify band intensities. A mouse monoclonal against β-actin (sc-69879, Santa Cruz, Dallas, TX, USA) was used as a loading control for Western blots and optical densities of each target protein normalized to β-actin. Horseradish peroxidase (HRP) conjugated anti-mouse or anti-rabbit IgG (Bio-rad) was used to detect the primary antibody. The ECL chemiluminescent detection system (PIERCE Inc., Thermofisher Scientific, Waltham, MA, USA) was used to detect the signals. Blots were imaged on the Fluorchem 8900 system (Alpha Innotech, San Leandro, CA, USA). The ratio of the intensity of dMBP/β-actin bands was quantified with NIH Image J software. The relative band intensity in AD samples was averaged and compared to the averaged band intensity of control samples.
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