Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology

Fluorescence two‐dimensional differential gel electrophoresis (2‐D DIGE * Difference Gel Electrophoresis (DIGE) technology is covered by US patent number 6043025 and foreign equivalents and patents pending. ©Amersham Pharmacia Biotech UK Limited, 2000 – All rights reserved. Amersham Pharmacia Biotech UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, England, U.K. HP7 9NA. Amersham Pharmacia Biotech AB, SE‐751 84 Uppsala Sweden. Amersham Pharmacia Biotech Inc, 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA. Amersham Pharmacia Biotech Europe GmbH , Munzinger Strasse 9, D‐791 11 Freiburg. Cy and ImageMaster are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc. Pharmacia is a trademark of Pharmacia Upjohn Inc. All goods and services are sold subject to terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and conditions are available on request. The inventors of the technology are Jon Minden and Alan Waggoner from the Carnegie Melon University. ) is a new development in protein detection for two‐dimensional gels. Using mouse liver homogenates (control and paracetamol (N‐acetyl‐p‐aminophenol, APAP)‐treated), we have determined the quantitative variation in the 2‐D DIGE process and established statistically valid thresholds for assigning quantitative changes between samples. Thresholds were dependent on normalised spot volume, ranged from approximately 1.2 fold for large volume spots to 3.5 fold for small volume spots and were not markedly affected by the particular cyanine dye combination or by multiple operators carrying out the dye labelling reaction. To minimise the thresholds, substantial user editing was required when using ImageMaster™ 2D‐Elite software. The difference thresholds were applied to the test system and quantitative protein differences were determined using replicate gels of pool samples and single gels from multiple individual animals (control vs treated in each gel). Throughout, the differences revealed with a particular cyanine dye combination were mirrored almost without exception when the dye combination was reversed. Both pool and individual sample analyses provided unique data to the study. The inter‐animal response variability in inbred mice was approximately nine times that contributed by the 2‐D DIGE process. A number of the most frequently observed protein change