Frozen colon samples were homogenized in PBS with 0.1% protease inhibitor cocktail (Sigma) and 1% phosphatase inhibitor cocktail (Sigma). Homogenates were centrifuged (12000 g, 15 min, 4 °C) and the supernatants were collected. Protein concentration was determined following Bradford’s colorimetric method. Aliquots of supernatants containing equal amounts of protein (25 μg) were separated on 4%-12% NuPAGE gel (Invitrogen) and then transferred to a nitrocellulose membrane (Hybond, GE Healthcare, United Kingdom). After blocking, membranes were incubated with COX-2 primary antibodies at the dilution of 1:500. After three washes, filter was then incubated with the secondary horseradish peroxidase linked anti-goat IgG. To check equal loading, the blots were analysed for β-actin expression. Immunodetection was performed using enhanced chemiluminiscence light-detecting kit (Amersham, Arlinghton Heights, IL, United States). Densitometric data were measured following normalisation to the control (house-keeping gene) Immunocomplexes were revealed by using the ECL detection system (GE Healthcare). Immunoblots were scanned with ImageScanner II (GE Healthcare) previously calibrated by using a greyscale marker (Kodak) and digitalized with Labscan 6.00 software (GE Healthcare).
Greyscale marker
Manufactured by Kodak
The Greyscale marker is a precision lab equipment designed for accurately marking and measuring grayscale tones. It features a range of distinct grayscale shades for precise calibration and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Imagescanner 2, by GE Healthcare (2 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
Hybond, by Cytiva (1 mentions)
Imagemaster 2d platinum v7, by GE Healthcare (1 mentions)
2 protocols using greyscale marker
1
Western Blot Analysis of COX-2 Expression
2
Quantitative Proteome Analysis of Colonic Tissue
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Silver stained gels were scanned with an ImageScanner II (GE Healthcare) previously calibrated by a greyscale marker (Kodak) and 2D images were captured with Labscan 6.00 software (GE Healthcare). Differential analysis was realized by using ImageMaster 2D Platinum v7.0 software (GE Healthcare) for spot detection, background subtraction, spot volume normalisation and differences in spot expression among the groups and/or each experiment. Each tissue sample was subjected to 2DE three times to minimize run-to-run variation, and each set of three gels was compared by using ImageMaster to confirm the nonappearance of statistically differential spots within the set of gels. The most representative silver-staining gels (gel migration, spot definition, and spot number) of each sample (n=5 per group) was used to identify differential colonic protein expression. The expression level was determined by the relative volume of each spot in the gel and expressed as %volume, calculated as spot volume/Σvolumes of all spots resolved in the gel. Variations in abundance, corresponding to normalized spot volume, were calculated as the ratio of average values of %volume for each group. Only spots with a %volume variation >1.4 were included in analysis. Statistical differences between the groups were determined using ANOVA (significance level P values < 0.05).
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