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    A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure

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    Authors
    Zhou, Shaobo
    Bailey, Matthew J.
    Dunn, Michael J.
    Preedy, Victor R.
    Emery, Peter W.
    Issue Date
    2005-08-01
    Subjects
    electrophoresis
    
    Metadata
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    Abstract
    We report the results of a systematic investigation to quantify the losses of protein during a well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([(14)C]bovine serum albumin and a homogenate prepared from the liver of a rat that had been injected with [(35)S]methionine) were used, and recovery was quantified by digesting pieces of gel in H(2)O(2) and subjecting the digests to liquid scintillation counting. When samples were loaded onto the first dimension immobilised pH gradient strips by in-gel rehydration, recovery of protein from the strips was 44-80% of the amount of protein loaded, depending on the amount of protein in the sample. Most of the unrecovered protein appeared to have adhered to the reswelling tray. Losses during isoelectric focusing (IEF) were much smaller (7-14%), although approximately 2% of the protein appeared to migrate from sample strips to adjacent blank strips in the focussing apparatus. A further 17-24% of the proteins were lost into the buffers during equilibration prior to running in the second dimension. Losses during the second dimension run and subsequent staining with SYPRO Ruby amounted to less than 10%. The overall loss during 2-DE was reduced by approximately 25% when proteins were loaded onto the IEF strips using sample cups instead of by in-gel rehydration. These extensive and variable losses during the 2-DE procedure mean that spot intensities on 2-DE gels cannot be used to derive reliable, quantitative information on the amounts of proteins present in the original sample.
    Citation
    Zhou S, Bailey MJ, Dunn MJ, Preedy VR, Emery PW (2005) 'A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure', Proteomics, 5 (11), pp.2739-2747.
    Publisher
    Wiley
    Journal
    Proteomics
    URI
    http://hdl.handle.net/10547/624606
    DOI
    10.1002/pmic.200401178
    PubMed ID
    15966007
    Additional Links
    https://onlinelibrary.wiley.com/doi/abs/10.1002/pmic.200401178
    Type
    Article
    Language
    en
    ISSN
    1615-9853
    ae974a485f413a2113503eed53cd6c53
    10.1002/pmic.200401178
    Scopus Count
    Collections
    Biomedical and biological science

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