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Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting

Nirmalan, NJ, Harnden, P, Selby, PJ and Banks, RE 2009, 'Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting' , The Journal of Pathology, 217 (4) , pp. 497-506.

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    Abstract

    The development of efficient formaldehyde cross-link reversal strategies will make the vast diagnostic tissue archives of pathology departments amenable to prospective and retrospective translational research, particularly in biomarker-driven proteomic investigations. Heat-induced antigen retrieval strategies (HIARs) have achieved varying degrees of crosslink reversal, potentially enabling archival tissue usage for proteomic applications outside its current remit of immunohistochemistry (IHC). While most successes achieved so far have been based on retrieving tryptic peptide fragments using shot-gun proteomic approaches, attempts at extracting full-length, non-degraded, immunoreactive proteins from archival tissue have proved challenging. We have developed a novel heat-induced antigen retrieval strategy using SDS-containing Laemmli buffer for efficient intact protein recovery from formalin-fixed tissues for subsequent analysis by western blotting. Protocol optimization and comparison of extraction efficacies with frozen tissues and current leader methodology is presented. Quantitative validation of methodology was carried out in a cohort of matched tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting for a selected panel of seven proteins known to be differentially expressed in renal cancer. Our data show that the protocol enables efficient extraction of non-degraded, fulllength, immunoreactive protein, with tumour versus normal differential expression profiles for a majority of the panel of proteins tested being comparable to matched frozen tissue controls (rank correlation, r = 0.7292, p < 1.825e-09). However, the variability observed in extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation of quantitative data from this subset of proteins. The method provides a viable, cost-effective quantitative option for the validation of potential biomarker panels through a range of clinical samples from existing diagnostic archives, provided that validation of the method is first carried out for the specific proteins under study.

    Item Type: Article
    Themes: Subjects outside of the University Themes
    Schools: Colleges and Schools > College of Science & Technology > School of Environment and Life Sciences > Ecosystems and Environment Research Centre
    Journal or Publication Title: The Journal of Pathology
    Publisher: Wiley
    Refereed: Yes
    ISSN: 00223417
    Depositing User: Users 29196 not found.
    Date Deposited: 20 Dec 2011 15:52
    Last Modified: 20 Aug 2013 18:19
    URI: http://usir.salford.ac.uk/id/eprint/19245

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