For Western blot analyses, cultured sciatic explants were prepared and thirty-five micrograms of total protein was separated and transferred to a nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA). The blots were reacted with the proper primary antibody (against GPRC5B or β-actin as control) and with horseradish peroxidase-conjugated secondary antibody. Detection was performed utilizing an enhanced chemiluminescence-Western blot system (Amersham Bioscience). LAS image-analysis system (Fujifilm, Tokyo, Japan) was used to quantification. All experiments were repeated a minimum of three times.
Las image analysis system
Manufactured by Fujifilm
Sourced in Japan
The LAS image analysis system is a comprehensive software platform designed for scientific image processing and analysis. It provides a range of tools for tasks such as image acquisition, enhancement, measurement, and quantification. The system offers a user-friendly interface and supports a variety of image file formats.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Cytiva (2 mentions)
Enhanced chemiluminescence western blot system, by Cytiva (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibodies, by Abmart (1 mentions)
Monoclonal antibody against β actin, by Merck Group (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Ecl advance kit, by Cytiva (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
4 protocols using las image analysis system
1
Quantitative Analysis of GPRC5B Expression
2
Western Blot Analysis of Protein Expression
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For Western immunoblotting, cultured cells were homogenized in lysis buffer (0.25 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% mercaptoethanol). The protein lysates were fractionated on a SDS-PAGE gel and transferred to nitrocellulose membrane (Millipore, USA). The blotted membranes were blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 hour, and the membranes were incubated overnight at 4°C with primary antibodies diluted in prepared TBST containing 3% nonfat milk. After 3 washes in TBST, the blots were reacted with horseradish peroxidase-conjugated secondary antibodies (Abmart, China) for 1 hour at room temperature and then washed again with TBST. For detection, an enhanced chemiluminescence-Western blot system (Amersham, Piscataway, USA) was used. For quantification, the X-ray films were put through a scanner (Samsung, Seoul, Korea) and analyzed with LAS image analysis system (Fujifilm, Tokyo, Japan). The picture were analysed with software ImageJ (National institutes of Health, USA) to obtain gray value of each band. Then relative gray value of each marker(gray value of special marker / gray value of β-actin)were calculated and analysed.
3
Western Blot Analysis of Aminoacyl-tRNA Synthetases
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Spinal dorsal horn (L4–5) tissue samples were homogenized in modified radioimmunoprecipitation assay buffer [RIPA; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% deoxycholic acid, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium o-vanadate, 1× protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Nutley, NJ, USA)]. After centrifugation for 30 minutes at 4°C, the supernatant was used for western blot analysis.
Western blot analysis was performed according to a previous study (Seo et al., 2014). We used the following primary antibodies: a polyclonal rabbit anti-FARSB (1:200; Proteintech, Chicago, IL, USA), a polyclonal rabbit anti-IARS (1:200; Neomics, Seoul, Korea), a polyclonal rabbit anti-MARS (Neomics, 1:200), a polyclonal rabbit anti-TARS (1:200; Gentex, San Antonio, TX, USA) and a monoclonal antibody against β-actin (1:5,000; Sigma). For quantification, images were scanned and analyzed with the LAS image analysis system (Fujifilm, Tokyo, Japan). Optical density (band intensity) ratios of FARSB, IARS, MARS and TARS to β-actin were calculated.
Western blot analysis was performed according to a previous study (Seo et al., 2014). We used the following primary antibodies: a polyclonal rabbit anti-FARSB (1:200; Proteintech, Chicago, IL, USA), a polyclonal rabbit anti-IARS (1:200; Neomics, Seoul, Korea), a polyclonal rabbit anti-MARS (Neomics, 1:200), a polyclonal rabbit anti-TARS (1:200; Gentex, San Antonio, TX, USA) and a monoclonal antibody against β-actin (1:5,000; Sigma). For quantification, images were scanned and analyzed with the LAS image analysis system (Fujifilm, Tokyo, Japan). Optical density (band intensity) ratios of FARSB, IARS, MARS and TARS to β-actin were calculated.
4
Western Blot Analysis of Sciatic Nerve Proteins
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For Western blot analysis, sciatic nerves were harvested and homogenized with a Polytron homogenizer in a modified radioimmune-precipitation assay buffer (150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.5% deoxycholic acid, 2 µg/mL aprotinin, 1 mM PMSF, 5 mM benzamidine, 1 mM sodium orthovanadate, and 1×protease inhibitor cocktail [Roche, Indianapolis, IN]). The lysates were centrifuged at 8,000g for 10 min at 4°C, and the supernatant was collected. Protein (25~35 μg) was separated by SDS-PAGE, and then transferred onto a nitrocellulose membrane (Amersham Biosciences). After blocking with 0.1% Tween-20 and 5% nonfat dry milk in Tris buffered saline (pH. 7.2, TBS) at room temperature for 1 h, the membranes were incubated with primary antibodies (1:500–1,000) in TBST containing 2% nonfat dry milk at 4°C overnight. After three 15 min washes with TBST, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:3,000) for 1 h at RT. The signals were detected using the ECL system (ECL Advance kit, Amersham Biosciences). For quantitative analysis, the films from 3 independent experiments (n = 3) were scanned and the intensity was analyzed using a LAS image analysis system (Fujifilm, Japan).
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