2D gel electrophoresis was performed on an Ettan DALT six electrophoresis unit (2 W per gel, 600 V, 400 mA; Amersham Biosciences; USA). The Cy2, Cy3 and Cy5 immunofluorescence was scanned using Typhoon 9400 scanner at the wavelength of 532 nm, 633 nm and 488 nm, respectively (Amersham Biosciences; USA). The digitalized images of the gels were analyzed using Image Master 2D Evolution version 2003.02 (Amersham Biosciences; USA). Spots in images were quantified and statistically evaluated using pattern analysis of computer-assisted pit assessment and the reproducibility and significance of the differences in spot sizes were determined.
Ettan daltsix electrophoresis unit
Manufactured by Cytiva
Sourced in United States, United Kingdom, Sweden
The Ettan DALTsix Electrophoresis Unit is a laboratory equipment designed for performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate and analyze proteins. It provides a platform for vertical protein separation based on molecular weight.
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3 protocols using ettan daltsix electrophoresis unit
1
2D Gel Electrophoresis Protein Analysis
2
Proteome Profiling of Plasma and Muscle
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Here, we briefly summarise the 2-DE procedure as the procedure was described in detail earlier [10 (link),23 (link),50 (link)]. Proteomic analysis of depleted plasma samples and prepared muscle biopsies was carried out using Ettan IPGphor 3 IEF System (GE Healthcare, Buckinghamshire, UK) (first dimension) and Ettan DALTsix Electrophoresis Unit (Amersham, Pharmacia, Uppsala, Sweden) (second dimension). The fluorescent stain SYPRO Ruby (Bio-Rad Laboratories, Hercules, CA, USA) was applied to plasma protein gels, and silver stain was applied to muscle biopsy gels. The stained protein pattern was visualized using a charge coupled device camera (VersaDoc Imaging system 4000 MP, Bio-Rad). The PDQuest Advanced (v. 8.0.1, Bio-Rad) software was used to analyse and quantify the protein pattern. The amount of protein in a certain spot was assessed as background corrected optical density integrated over all pixels in the spot and expressed as integrated optical density (IOD). The parts per million (ppm) values for all proteins were generated and used for further statistical analysis.
3
2-D Gel Electrophoresis for Proteomic Analysis
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Here, we briefly summarise the 2-DE procedure as the procedure has been described in detail earlier. [16, 28, 37] Depleted plasma samples containing 100 mg total protein were run in the first dimension. This was followed by second dimension separation using Ettan DALTsix Electrophoresis Unit (Amersham, Pharmacia, Uppsala, Sweden). The protein gels were fluorescently stained with SYPRO Ruby (Bio-Rad Laboratories, Hercules, CA). We visualized the stained protein pattern using a charge coupled device camera (VersaDoc Imaging system 4000 MP, Bio-Rad) and further analyzed and quantified the protein pattern using PDQuest Advanced (v. 8.0.1, Bio-Rad). The amount of protein in a certain spot was assessed as background corrected optical density, integrated over all pixels in the spot and expressed as integrated optical density. The parts per million values for all proteins were generated and were evaluated for differences between the groups using MVDA. Two preparative gels (1 pool from CWP and 1 from CON, containing 400 mg of total protein) for protein identifications were run according to the above described protocol.
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