Abstract
The performance of Cibacron Blue dye (HiTrapBlue or Affigel Blue) in depleting albumin from plasma, as a pre-treatment for biomarker searching in the low-abundance proteome, is here assessed. It is shown that (i) co-depletion of non-albumin species is an ever-present hazard; (ii) the only proper eluant able to release quantitatively the proteins bound to the dye is boiling 4% SDS-25mM DTT, an ion shock (2M NaCl) being quite ineffective in releasing the low-abundance species tightly bound to the dye moiety; (iii) the mechanism of dye-protein interaction, after an initial ion-ion docking, is a robust hydrophobic interaction, which progressively augments at lower and lower pH values; (iv) at pH 2.2 in the presence of 0.1% TFA, the blue resin behaves, for all practical purposes, just as a reverse-phase chromatography column, since all residual proteins present in plasma are completely harvested. However Cibacron Blue technology should not necessarily be discarded: As long as also the plasma fraction adsorbed is properly released and analyzed, together with the flow through, one should be able to perform a viable analysis of the low-abundance proteome.
Original language | English |
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Pages (from-to) | 3638-3644 |
Number of pages | 7 |
Journal | Electrophoresis |
Volume | 32 |
Issue number | 24 |
DOIs | |
Publication status | Published - Dec 2011 |
Keywords
- Albumin depletion
- Biomarkers
- Cibacron blue
- Elution protocols
- Plasma
ASJC Scopus subject areas
- Biochemistry
- Clinical Biochemistry