Department of BiochemistrySchool of Medicine & Biomedical Sciences
2D-DIGE
Department of Biochemistry
A complete PDF manual describing DIGE technology can be viewed here: Ettan DIGE Manual. The sections of this manual include an introduction to the technology and experimental design considerations, protocols for sample preparation and Cy-dye labeling, detailed instructions for 2D-electrophoresis and gel scanning, use of Decyder software for quantification, and automated spot picking.
2-D DIGE technology is available on a user fee or fee for service basis to all investigators. Investigators are expected to prepare and quantify their own lysates, and should refer to the DIGE manual for specific protocols. Note that it is essential that lysis buffers be free of both primary amines and sulfhydryl groups. Downstream of this, technologies available in the facility include: Cy-dye labeling, isoelectric focusing using Immobilized pH Gradient strips, 2nd dimension SDS gel electrophoresis, fluorescence scanning, image analysis with the Decyder 2D software package, and automated excision of desired spots.
Fees
2-D DIGE fees are structured on a two-tier level: user fees and fee for service. User fees cover the costs of consumables and the maintenance of facility instrumentation. Under this fee structure, users are provided all necessary materials and guided through the process of labeling their cell extracts, running 1st and 2nd dimension separations, use of the fluorescence scanner, given initial training in the use of the Decyder 2D software, and assisted in the use of the automated spot picker.
Investigators may also choose to make use of the facility on a fee for service basis. In this case, upon receiving lysates of known total protein concentration in buffers suitable for Cy-dye labeling, the facility will perform 2-D DIGE with analysis via the Decyder 2-D software and provide the investigator with quantitative data on up to 30 proteins of interest, as well as provide the corresponding gel plugs for further analysis if desired. Prospective 2-D DIGE users must meet with the facility director and manager to discuss experimental design and expectations prior to use of the facility. Investigators should contact the facility manager, Sujith Alphy (829-2215, 829-2159; alphy@buffalo.edu), to arrange pre-use consultations.
2 Dye Gel (Cy-labeling/1st & 2nd Dimension Separation/Image Scanning) 3 Dye Gel (Cy-labeling/1st & 2nd Dimension Separation/Image Scanning) Quantitative Image Analysis with Decyder 2-D Sypro Staining & Automated Spot Picking Fees are mandatory: all P.I.'s wishing to use the facility instrumentation will be charged. However, upon consultation with the facility director, Dr. Kenneth Blumenthal (829-2727; kblumen@buffalo.edu), unfunded investigators may be granted limited access to the facility for the purpose of obtaining preliminary data in support of grant applications.
Post-DIGE analysis of Proteins of Interest
MALDI-TOF MS is available for identification of unknown proteins via mass analysis of proteolytic peptides. Investigators wishing to do their own analysis of proteins obtained via-2D-DIGE should refer to the MALDI-TOF portion of this website for instrument and technical information.
Analysis of proteins obtained via-2D-DIGE is also available on a fee for service basis. Fees for MALDI-TOF analysis by facility staff are:1-5: $40/sample; each additional sample, up to 10: $30/sample; additional samples up to 15: $20/sample. Investigators wishing to analyze greater than 15 unknown proteins should consult with facility staff about the use of available high-throughput automated technologies.
Useful link: Proteomics ProtocolsLink: back to Core Research Facilities
Link: MALDI - TOF MS
Link: BIAcore X