Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics

Nature Protocols volume 1, pages 2940–2956 (2006)Cite this article

This article has been updated

Abstract

Proteome data combined with histopathological information provides important, novel clues for understanding cancer biology and reveals candidates for tumor markers and therapeutic targets. We have established an application of a highly sensitive fluorescent dye (CyDye DIGE Fluor saturation dye), developed for two-dimensional difference gel electrophoresis (2D-DIGE), to the labeling of proteins extracted from laser microdissected tissues. The use of the dye dramatically decreases the protein amount and, in turn, the number of cells required for 2D-DIGE; the cells obtained from a 1 mm2 area of an 8–12 μm thick tissue section generate up to 5,000 protein spots in a large-format 2D gel. This protocol allows the execution of large-scale proteomics in a more efficient, accurate and reproducible way. The protocol can be used to examine a single sample in 5 d or to examine hundreds of samples in large-scale proteomics.

NOTE: In the PDF version of this article initially published online, the publication date was shown as 25 January 2006 instead of 25 January 2007. The error has been corrected in the PDF version of the article.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Timetable showing the flow of the experimental protocol described, employing laser microdissection, 2D-DIGE and in-gel digestion in cancer proteomics.
Figure 2: Example of a laboratory equipped with the range of 2D-DIGE devices required for large-scale proteomics.
Figure 3: Appearance of a gradient-gel making system.
Figure 4: Dark box used for gel storage.
Figure 5: Typical 2D images of the proteins extracted from a 1 mm2 area of (a) liver cancer tissue and (b) lung adenocarcinona tissue.

Similar content being viewed by others

Change history

  • 22 February 2007

    changed 2006 to 2007

References

  1. Espina, V. et al. Laser-capture microdissection. Nat. Protoc. 1, 586–603 (2006).

    Article  CAS  Google Scholar 

  2. Buckanovich, R.J. et al. Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment. Cancer Biol. Ther. 5, 635–642 (2006).

    Article  CAS  Google Scholar 

  3. Banks, R.E. et al. The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis—preliminary findings. Electrophoresis 20, 689–700 (1999).

    Article  CAS  Google Scholar 

  4. Gorg, A., Weiss, W. & Dunn, M.J. Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685 (2004).

    Article  Google Scholar 

  5. Carrette, O., Burkhard, P.R., Sanchez, J.C. & Hochstrasser, D.F. State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research. Nat. Protoc. 1, 812–823 (2006).

    Article  CAS  Google Scholar 

  6. Oh, J.M. et al. A database of protein expression in lung cancer. Proteomics 1, 1303–1319 (2001).

    Article  CAS  Google Scholar 

  7. Craven, R.A., Totty, N., Harnden, P., Selby, P.J. & Banks, R.E. Laser capture microdissection and two-dimensional polyacrylamide gel electrophoresis: evaluation of tissue preparation and sample limitations. Am. J. Pathol. 160, 815–822 (2002).

    Article  CAS  Google Scholar 

  8. Bravo, R., Bellatin, J. & Celis, J.E. [35S]-methionine labelled polypeptides from HELA cells. Coordinates and percentage of some major polypeptides. Cell Biol. Int. Rep. 5, 93–96 (1981).

    Article  CAS  Google Scholar 

  9. Poznanovic, S. et al. Differential radioactive proteomic analysis of microdissected renal cell carcinoma tissue by 54 cm isoelectric focusing in serial immobilized pH gradient gels. J. Proteome Res. 4, 2117–2125 (2005).

    Article  CAS  Google Scholar 

  10. Kondo, T. et al. Application of sensitive fluorescent dyes in linkage of laser microdissection and two-dimensional gel electrophoresis as a cancer proteomic study tool. Proteomics 3, 1758–1766 (2003).

    Article  CAS  Google Scholar 

  11. Unlu, M., Morgan, M.E. & Minden, J.S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077 (1997).

    Article  CAS  Google Scholar 

  12. Shaw, J. et al. Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes. Proteomics 3, 1181–1195 (2003).

    Article  CAS  Google Scholar 

  13. Lee, J.R. et al. Differential protein analysis of spasomolytic polypeptide expressing metaplasia using laser capture microdissection and two-dimensional difference gel electrophoresis. Appl. Immunohistochem. Mol. Morphol. 11, 188–193 (2003).

    Article  CAS  Google Scholar 

  14. Zhou, G. et al. 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol. Cell Proteomics 1, 117–124 (2002).

    Article  CAS  Google Scholar 

  15. Alban, A. et al. A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36–44 (2003).

    Article  CAS  Google Scholar 

  16. Uwakwe, C.J. CyDye DIGE Fluor saturation dyes for labelling and detection of scarce protein samples using Ettan DIGE system: application of the pooled internal standard to the investigation of a model system. Amersham Biosciences Life Science News 16, 1–4 (2004).

    Google Scholar 

  17. Uhlen, M. & Ponten, F. Antibody-based proteomics for human tissue profiling. Mol. Cell Proteomics 4, 384–393 (2005).

    Article  CAS  Google Scholar 

  18. Okano, T. et al. Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE). Proteomics 6, 3938–3948 (2006).

    Article  CAS  Google Scholar 

  19. Tan, H.T., Zubaidah, R.M., Tan, S., Hooi, S.C. & Chung, M.C. 2-D DIGE analysis of butyrate-treated HCT-116 cells after enrichment with heparin affinity chromatography. J. Proteome Res. 5, 1098–1106 (2006).

    Article  CAS  Google Scholar 

  20. Ahmed, N. & Rice, G.E. Strategies for revealing lower abundance proteins in two-dimensional protein maps. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 815, 39–50 (2005).

    Article  CAS  Google Scholar 

  21. Lescuyer, P., Hochstrasser, D.F. & Sanchez, J.C. Comprehensive proteome analysis by chromatographic protein prefractionation. Electrophoresis 25, 1125–1135 (2004).

    Article  CAS  Google Scholar 

  22. Qin, S. et al. Chromatofocusing fractionation and two-dimensional difference gel electrophoresis for low abundance serum proteins. Proteomics 5, 3183–3192 (2005).

    Article  CAS  Google Scholar 

  23. Young, D.A., Voris, B.P., Maytin, E.V. & Colbert, R.A. Very-high-resolution two-dimensional electrophoretic separation of proteins on giant gels. Meth. Enzymol. 91, 190–214 (1983).

    Article  CAS  Google Scholar 

  24. Klose, J. et al. Genetic analysis of the mouse brain proteome. Nat. Genet. 30, 385–393 (2002).

    Article  CAS  Google Scholar 

  25. Seike, M. et al. Proteomic signatures for histological types of lung cancer. Proteomics 5, 2939–2948 (2005).

    Article  CAS  Google Scholar 

  26. Fujii, K. et al. Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dye. Proteomics 5, 1411–1422 (2005).

    Article  CAS  Google Scholar 

  27. Fujii, K. et al. Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye. Proteomics 6, 1640–1653 (2006).

    Article  CAS  Google Scholar 

  28. Suehara, Y. et al. Proteomic signatures corrsponding to histological classification and grading of soft-tissue sarcomas. Proteomics 6, 4402–4409 (2006).

    Article  CAS  Google Scholar 

  29. Hatakeyama, H. et al. Protein clusters associated with carcinogenesis, histological differentiation and nodal metastasis in esophageal cancer. Proteomics 6, 6300–6317 (2005).

    Article  Google Scholar 

  30. Rhodes, D.R. & Chinnaiyan, A.M. Integrative analysis of the cancer trasncriptome. Nat. Genet. 37, S31–S37 (2005).

    Article  CAS  Google Scholar 

  31. Chen, G. et al. Protein profiles associated with survival in lung adenocarcinoma. Proc. Natl Acad. Sci. USA 100, 13537–13542 (2003).

    Article  CAS  Google Scholar 

  32. Greengauz-Roberts, O. et al. Saturation labeling with cysteine-reactive cyanine fluorescent dyes provides increased sensitivity for protein expression profiling of laser-microdissected clinical specimens. Proteomics 5, 1746–1757 (2005).

    Article  CAS  Google Scholar 

  33. Wilson, K.E. et al. Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling. Proteomics 5, 3851–3858 (2005).

    Article  CAS  Google Scholar 

  34. Sitek, B. et al. Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma. Proteomics 5, 2665–2679 (2005).

    Article  CAS  Google Scholar 

  35. Sitek, B. et al. Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling. Proteomics 6, 4337–4345 (2006).

    Article  CAS  Google Scholar 

  36. GE Healthcare Bio-Sciences. Detection and mass spectrometry identification of protein changes in low-abundance tissue using CyDye DIGE Fluor saturation dyes. Application note 18117751AA, 2003.10, 1–8.

  37. Rabilloud, T., Valette, C. & Lawrence, J.J. Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 15, 1552–1558 (1994).

    Article  CAS  Google Scholar 

  38. Sanchez, J.C. et al. Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 18, 324–327 (1997).

    Article  CAS  Google Scholar 

  39. Sapan, C.V., Lundblad, R.L. & Price, N.C. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29, 99–108 (1999).

    CAS  PubMed  Google Scholar 

  40. Maeda, K., Finnie, C. & Svensson, B. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms. Biochem. J. 378, 497–507 (2004).

    Article  CAS  Google Scholar 

  41. Fujii, K. et al. Protein expression pattern distinguishes different lymphoid neoplasms. Proteomics 5, 4274–4286 (2005).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Proteome Bioinformatics Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Tadashi Kondo & Setsuo Hirohashi

Authors
  1. Tadashi Kondo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Setsuo Hirohashi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tadashi Kondo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kondo, T., Hirohashi, S. Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics. Nat Protoc 1, 2940–2956 (2006). https://doi.org/10.1038/nprot.2006.421

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.421

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing