Protein Extraction from Solid Tissue

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 675)

Abstract

Maximal extraction and solubilization of protein from diseased or healthy tissue is important to make the whole protein complement available for proteomic analysis. It also helps to maximize reproducibility and to minimize waste. Minimal degradation of the protein amino acid backbone or dephosphorylation is essential to preserve the analytical utility of the extract. Containment of the sample is important to minimize the risk of contamination to and from the sample. The proposed standard protocol for protein extraction and solubilization can result in 98% solubilization of brain tissue, corresponding to about 100 μg protein per mg tissue wet weight, by a frozen disintegration/SDS-based solubilization method: Tissue is crushed in the frozen state in a cryotube by shaking with a sterile steel ball. The crushing is followed by the extraction and solubilization in 2% SDS for 10 min, at 70°C, in a volume corresponding to ten times the tissue wet weight, with shaking. The containment in a cryotube helps to prevent contamination. The treatment with SDS sample buffer can inhibit protease and phosphatase activity. The resulting protein extracts can be used for SDS PAGE, 2-D PAGE, Western blotting, ESI-MS, and ELISA. The proposed standard protocol has the potential to find wide application where protein extraction, solubilization, identification, and quantitation from cryopreserved clinical samples are desirable.

Key words

Protein Proteome Phosphorylation Extraction Solubilization Tissue Analysis Identification Quantitation 

Notes

Acknowledgment

The authors would like to thank the Wallenberg Consortium North for support.

References

  1. 1.
    Ericsson, C., B. Franzén, and M. Nistér, (2006) Frozen tissue biobanks; Tissue handling, cryopreservation, extraction and use for proteomic analysis. Acta Oncologica, 45, 643–61.PubMedCrossRefGoogle Scholar
  2. 2.
    Scopes, R.K., (1987) Making an Extract, in Protein Purification: Principles and Practice. Springer-Verlag: New York.Google Scholar
  3. 3.
    Ericsson, C., I. Peredo, and M. Nistér, (2007) Optimized protein extraction from cryopreserved brain tissue samples. Acta Oncologica, 46(1), 10–20.PubMedCrossRefGoogle Scholar
  4. 4.
    Butt, R.H. and J.R. Coorssen, (2006) Pre-extraction sample handling by automated ­frozen disruption significantly improves sub­sequent proteomic analyses. Journal of Proteome Research, 5(2), 437–48.PubMedCrossRefGoogle Scholar
  5. 5.
    Olivieri, E., B. Herbert, and P.G. Righetti, (2001) The effect of protease inhibitors on the two-dimensional electrophoresis pattern of red blood cell membranes. Electrophoresis, 22(3), 560–5.PubMedCrossRefGoogle Scholar
  6. 6.
    Rai, A.J., C.A. Gelfand, B.C. Haywood, D.J. Warunek, J. Yi, M.D. Schuchard, et al., (2005) HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples. Proteomics, 5(13), 3262–77.PubMedCrossRefGoogle Scholar
  7. 7.
    Laemmli, U.K., (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(259), 680–5.PubMedCrossRefGoogle Scholar
  8. 8.
    Laemmli, U.K. and M. Favre, (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. Journal of Molecular Biology, 80(4), 575–99.PubMedCrossRefGoogle Scholar
  9. 9.
    Bjellqvist, B., K. Ek, P.G. Righetti, E. Gianazza, A. Gorg, R. Westermeier, et al., (1982) Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. Journal of Biochemical and Biophysical Methods, 6(4), 317–39.PubMedCrossRefGoogle Scholar
  10. 10.
    Peng, J., J.E. Elias, C.C. Thoreen, L.J. Licklider, and S.P. Gygi, (2003) Evaluation of multidimensional chromatography coupled with ­tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. Journal of Proteome Research, 2(1), 43–50.PubMedCrossRefGoogle Scholar
  11. 11.
    Nielsen, U.B., M.H. Cardone, A.J. Sinskey, G. MacBeath, and P.K. Sorger, (2003) Profiling receptor tyrosine kinase activation by using Ab microarrays. Proceedings of the National Academy of Sciences of the United States of America, 100(16), 9330–5.PubMedCrossRefGoogle Scholar
  12. 12.
    Harder, A., R. Wildgruber, A. Nawrocki, S.J. Fey, P.M. Larsen, and A. Gorg, (1999) Comparison of yeast cell protein solu­bilization procedures for two-dimensional ­electrophoresis. Electrophoresis, 20(4–5), 826–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Oncology-PathologyKarolinska InstitutetStockholmSweden

Personalised recommendations