Difference Gel Electrophoresis (DIGE) Using CyDye DIGE Fluor Minimal Dyes

Bulbul Chakravarti1, Sean R. Gallagher2, Deb N. Chakravarti1

1 Keck Graduate Institute of Applied Life Sciences, Claremont, California, 2 UVP, Inc., Upland, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.23
DOI:  10.1002/0471142727.mb1023s69
Online Posting Date:  February, 2005
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One‐ and two‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1‐ and 2‐D SDS‐PAGE) have been widely used for the separation and quantitative estimation of proteins. Following electrophoresis, the gels are stained appropriately to visualize the proteins. Difference gel electrophoresis (DIGE) is a new technique in which different protein samples, individually labeled with specific CyDyes, are combined together followed by electrophoresis and post electrophoretic co‐detection and co‐analysis on the same gel. CyDye DIGE fluor minimal dyes, which consist of three different CyDyes with different spectral characteristics, have been widely used for such purposes. The technique is highly sensitive with a wide dynamic range for detection of proteins and compatible with state‐of‐the‐art protein identification techniques using mass spectrometry. Although DIGE is mainly used to compare differential expression of various protein samples using 2‐D SDS‐PAGE, 1‐D DIGE also has important applications in quantitative proteomic studies.

Keywords: one‐dimensional DIGE; proteomics; CyeDye DIGE fluor minimal dyes

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Table of Contents

  • Basic Protocol 1: Labeling of Proteins with CyDye Dige Fluor Minimal Dyes
  • Support Protocol 1: Preparation of Sample for Labeling
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Labeling of Proteins with CyDye Dige Fluor Minimal Dyes

  • CyDye DIGE fluor minimal dyes (store at −20°C in dark)
  • Dimethyl formamide (DMF)
  • Protein sample (see protocol 2)
  • 10 mM lysine
  • 2× gel loading buffer (see recipe)
  • 5% to 15% (w/v) acrylamide gradient gel (unit 10.2)
  • Low‐fluorescence glass plates (Amersham Biosciences)
  • Typhoon variable mode imager 9400 series (Amersham Biosciences) or charge‐coupled device (CCD)–based imaging system (AC1 Autochemisystem from UVP) with UV transilluminator (UVP)
  • ImageQuant software (Amersham Biosciences) or similar software
  • Additional reagents and equipment for 1‐D SDS‐PAGE (unit 10.2)

Support Protocol 1: Preparation of Sample for Labeling

  • Cells of interest: 20 to 50 ml of microbial culture medium (e.g., Escherichia coli, ∼1 × 108 cells/ml), 20 to 50 ml of tissue culture medium for mammalian cells (∼5 × 105 cells/ml), or ∼200 mg of tissue
  • Cell wash buffer (see recipe)
  • Lysis buffer (see recipe)
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Literature Cited

Literature Cited
   Chakravarti, D.N., Russell, D.P., Wooters, J.L., Greene, B.A., Masi, A.W., and Zagursky, R.J. Novel Streptococcus pneumoniae Open Reading Frames Encoding Polypeptide Antigens and Uses Thereof. International patent application number WO 02/083855, published October 24, 2002.
   Gade, D., Thiermann, J., Markowsky, D., and Rabus, R. 2003. Evaluation of two‐dimensional difference gel electrohoresis for protein profiling. J. Mol. Microbiol. Biotechnol. 5:240‐251.
   Gharbi, S., Gaffney, P., Yang, A., Zvelebil, M.J., Cramer, R., Waterfield, M.D., and Timms, J.F. 2002. Evaluation of two‐dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system. Mol. Cell. Proteomics 1:91‐98.
   Patton, W.F. 2002. Detection technologies in proteome analysis. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 771:3‐31.
   Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J., Pognan, F., Hawkins, E., Currie, I., and Davison, M. 2001. Validation and development of fluorescence two‐dimensional differential gel electrophoresis proteomics technology. Proteomics 1:377‐396.
   Ünlü, M., Morgan, M.E., and Minden, J.S. 1997. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 18:2071‐2077.
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