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

  • Shaobo Zhou
    ,
  • Matthew J. Bailey
    ,
  • Michael J. Dunn
    ,
  • Victor R. Preedy
    ,
  • Peter W. Emery
  • King's College London
    ,
  • University College Dublin
Research Output: Contribution to journal Article Peer-review

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.

Publication Information

Output type

Research Output: Contribution to journal Article Peer-review

Original language

English

Pages from-to (Number of pages)

Pages 2739-2747

Journal (Volume, Issue Number)

Proteomics (Volume 5, Issue 11)

Publication milestones

  • Published - 01/08/2005

Publication status

Published - 01/08/2005

ISSN

1615-9853

External Publication IDs

  • handle.net: 10547/624606
  • Scopus: 23044464125

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