Services are available to non-profit institutions within Delaware. Other partner institutions and institutions within the National INBRE/COBRE network may also apply for service.
Please consult with the core facility before preparing samples.
To identify changes in protein expression, we use DIGE technology. In DIGE analysis, protein samples are trace labeled with charge- and mass-matched fluorescent dyes. Typically, a set of control samples are labeled with one dye and a set of treated samples are labeled with a second dye. A standards sample is prepared by combining control and treated samples and labeling with a third dye. Individual control and treated samples are combined with a common standard sample and proteins separated by 2D gel electrophoresis. The standard sample is used to enable comparison of multiple gels using DIGE software. With 4 or more samples, statistical analysis is used to determine protein spots that differ significantly between the control and treated samples. Protein spots of interest are picked from the gel for trypsin digestion and mass analysis. Using the DIGE system, only proteins that change significantly need to be identified by mass analysis whereas in shot-gun isobaric tag proteomics all peptides have to be analyzed by MS/MS to identify proteins by peptide sequence. DIGE analysis is labor intensive at the front end while shot-gun proteomics is mass spec intensive. We hope to be able to help investigators with isobaric tag proteomics in the near future.
1. Define choice of samples
2. optimize solubilization of proteins
3. optimize separation of proteins
4. identify differentially expressed protein spots
5. pick spots, digest and MS/MS for identification of protein spots