HMVEC-L cells were harvested by centrifugation and then disrupted with lysis buffer containing 5 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% Triton X-100, and 2 mM PMSF. The cell lysate was centrifuged at 12,000 Χ g for 30 min, and the supernatant fraction was collected. Protein concentrations were determined using a BCA assay kit (Pierce). Immobiline DryStrips (Amersham Biosciences) were used for isoelectric focusing, which was carried out with 1 mg of the extracted protein on an IPGphor system (Amersham Biosciences). After IEF separation, the proteins were separated in the second dimension by SDS-PAGE. For image analysis, the gels were visualized with Coomassie Brilliant Blue G-250 according to the manufacturer’s instructions. The 2-D gels were scanned with an ImageScanner (Amersham Biosciences) in transmission mode. Spot detection and matching were performed using ImageMaster 2D version 5.0 (Amersham Biosciences). Digitized images were analyzed using the ImageMaster program to calculate the 2-D spot intensity by integrating the optical density over the spot area (the spot “volume”) and normalized. The values were normalized and then exported to SPSS 18.0 for statistical analysis.
Imagemaster 2d version 5
Manufactured by Cytiva
ImageMaster 2D version 5.0 is a software application for the analysis and quantification of two-dimensional gel electrophoresis images. The core function of this software is to assist researchers in the identification, quantification, and comparison of protein spots in 2D gel images.
Automatically generated - may contain errors
2 protocols using imagemaster 2d version 5
1
Quantitative Proteomics Profiling of HMVEC-L Cells
2
Two-Dimensional Gel Electrophoresis of Lung Proteins
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Protein (200 µg) from each group of lung tissue was used for the 2DE analysis. First, 1 mg of protein from each lung sample was precipitated with 10% trichloroacetic acid in acetone and resuspended in the sample solution. Immobiline Dry Strips (Amersham Biosciences, Little Chalfont, UK) were used for isoelectric focusing, which was carried out using 1 mg of the extracted protein in a MultiPhor II system (GE Healthcare, Little Chalfont, UK). After isoelectric focusing separation, the proteins were separated in the second dimension by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For image analysis, the gels were visualized with Coomassie Brilliant Blue G-250 according to the manufacturer's instructions. The 2D gels were scanned with an ImageScanner (Bio-Rad Laboratories Inc., Hercules, CA, USA) in transmission mode. Spot detection and matching were performed using ImageMaster 2D version 5.0 (Amersham Biosciences). Digitized images were analyzed using the ImageMaster software to calculate the 2D spot intensity by integrating the optical density over the spot area (the spot "volume"); the images were then normalized [19 (link)].
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