| Overview |
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| | The Difference Gel Electrophoresis (DiGE) system is a specialized two-dimensional electrophoresis technique that uses three different fluorescent dyes (currently Cy-3, Cy-5, and Cy2) to label the two protein samples being compared. The fluorescent dyes have been designed so that they do not alter the charge of proteins or impart significant electrophoretic mobility differences to identical proteins. The gel is scanned at the wavelengths corresponding to the dyes used. Thus, in DiGE, every protein in one sample superimposes with its differentially labeled counterpart in the second sample. By overlaying the two images, it is easy to visualize those proteins that differ between the two samples. Compared to traditional 2-D gel techniques for protein profiling, DiGE provides a significantly more efficient, sensitive, and reliable way to detect proteins whose expression is altered between control and treated samples.
After imaging analysis, those differentiated protein spots can be cut off from the gel by the integrated picker. Washing and proteolytic digestion are performed prior to the mass spectrometry analysis. MS analysis is commonly performed using Peptide Mass Fingerprinting (PMF). Peptide sequences can also be further analyzed by MS/MS.
Traditional 2D gel electrophoresis, along with several staining method are also available on request. Staining methods include: Coomassie Brilliant Blue® staining, silver staining, Sypro® ruby staining or other post-staining methods. These staining methods vary in sensitivity and compatibility for downstream proteolytic digestion and mass spectrometry. |
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| About the Facility |
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| | The 2D gel electrophoresis laboratory is located on the 9th floor of BST3. The facility uses BioRad® first and second dimension electrophoresis equipment and supplies. The DiGE scanner and integrated picker is a custom built instrument with a high-resolution cooled Prometrix® CCD camera capable of imaging Cy2, Cy3 and Cy5 dyes. Currently, imaging analysis is done by using DeCyder® Differential Analysis software (Amersham Biosciences Inc.) and V++® Precision Digital Imaging System (Digital Optics Ltd). If you are interested in utilizing the services of this laboratory, please contact Paul Wood at: 412-648-9618, jal18@pitt.edu. |
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| Process |
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| | 2-D gel electrophoresis process consists of these steps:
1. Sample preparation
2. First dimension isoelectric focusing
3. Second dimension gel electrophoresis
4. Staining
5. Imaging analysis
6. Spots excision |
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| Forms |
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| | Please see our Forms page |
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| Pricing |
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| | Pricing for internal users as well as users outside the University of Pittsburgh are available in pdf format as a complete Price List of services provided by the GPCL. |
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| Frequently Asked Questions |
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| | Coming soon |
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| Policies |
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| | Confidentiality
The staff of the facility will maintain strict confidentiality of the research projects for the investigators using the facility. No data for a given investigator will be accessible to anyone but the PI or their designated contacts.
Facility Acknowledgement
The Genomics and Proteomics Core Laboratories request an acknowledgement in the Methods section of any publications resulting from the data produced within the facility. An example is "Difference Gel electrophoresis was performed by the Genomics and Proteomics Core Laboratories at the University of Pittsburgh." We request that a reference for of scientific papers be forwarded to the GPCL Administrator for all publications that come from our work. Thank you for your consideration of this important request.
Support Letter
The Scientific Director will gladly provide investigators with a letter of support stating our willingness to assist you with your research. Please contact the GPCL Administrator, Tracy Crawford 412-648-9787.
Billing
For internal users, billing will be done automatically by interdepartmental charges (IDC). Users outside the university will receive an invoice by mail and are required to provide partial payment prior to beginning work.
Approvals
All projects using animal or human tissues must have an approved protocol with the IACUC or IRB.
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| Sample Submission Guidelines |
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| | Please refer to this form. |
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| Shipping and Scheduling |
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| | If samples will be shipped to the GPCL then please arrange in advance for the shipping by contacting Paul Wood (412-648-9618); email
Shipping address
University of Pittsburgh
Attn: GPCL Proteomics Laboratories
9035 Biomedical Science Tower 3
3501 Fifth Avenue
Pittsburgh, PA 15260
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