Purification of human serum amyloid A by anion-exchange fast protein liquid chromatography

Hocke, Gerti; Kaffarnik, H; Münscher, Gerhard; Steinmetz, Armin

doi:10.1016/s0378-4347(00)82499-8

Cited by 11 publications

(1 citation statement)

References 21 publications

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“…Purification of SAA may be of interest for several purposes such as molecular investigations of SAA [10,11], investigation of the kinetics [5] and functions of SAA [12], investigation of the pathogenesis of amyloidosis [13], or production of standards for SAA assays [14]. However, SAA tends to precipitate [13] or form large protein aggregates [15] when it is separated from HDL, and consequently, large-scale purification of SAA is a challenging discipline [16].…”

Section: Resultsmentioning

confidence: 99%

Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum – a pilot study

et al. 2013

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BackgroundSerum amyloid A (SAA) is useful as a diagnostic marker of systemic inflammation in horses, but only heterologous assays based on non-equine calibration and standardization are available for measurements of equine SAA. More accurate measurements could be obtained using purified species-specific SAA in native conformation for assay calibration and standardization. Further knowledge about the biochemical properties of SAA would facilitate a future production of native species-specific calibration material Therefore, the aim of the study was an investigation of the solubility and potentials for purification of equine SAA based on biochemical properties.Freeze dried equine acute phase serum was dissolved in 70% 2-propanol, 8 M urea, and milli-Q water, respectively. Supercritical fluid extraction (SFE), size-exclusive chromatography (FPLC-SEC), and preparative isoelectric focusing (IEF) were performed in the attempt to purify. Immunostaining of IEF blots were used for isoform-specific detection of SAA in the preparations and purity was assessed by silverstained SDS-PAGE.FindingsSAA was soluble in 70% 2-propanol, 8 M urea and Milli-Q water. SAA was not separated in the lipophilic or ampipathic fractions following SFE. SAA was included in a FPLC-SEC-fraction of 237 kDa, despite the molecular weight known to be much smaller, suggesting binding to other serum constituents. SAA precipitated following separation of other serum proteins by preparative IEF.DiscussionNo effective purification of SAA was achieved in the present study, but findings important for future investigations were made. The study suggested that SAA is not exclusively hydrophobic, but appears less hydrophobic when interacting with other serum components. These results suggest more complex aspects of solubility than previously believed, and indicate potentials for purification of native SAA.

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Section: Resultsmentioning

confidence: 99%

Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum – a pilot study

et al. 2013

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Chromatography of Membrane Proteins and Lipoproteins

Zolla¹

2000

Encyclopedia of Analytical Chemistry

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The available methods for the separation of membrane proteins and lipoproteins are sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, isoelectric focusing (IEF), capillary electrophoresis (CE) and high‐performance liquid chromatography (HPLC). In this article it is shown that HPLC techniques, given their wide versatility, relative ease of use, and high resolution, may be considered the most valuable tool for the characterization of virtually any hydrophobic protein. Moreover, HPLC is not a destructive technique and therefore proteins, once separated, are available for further analytical investigations. Application examples are described and comparisons with other methods are discussed.

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Chromatography of Membrane Proteins and Lipoproteins

Zolla

D’Alessandro

2011

Encyclopedia of Analytical Chemistry

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The available methods for the separation of membrane proteins and lipoproteins are sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), followed by immunoblotting, isoelectric focusing (IEF), and capillary electrophoresis (CE), along with the recently introduced gel‐based native techniques (blue native (BN) and clear native (CN)), and high‐performance liquid chromatography (HPLC). In this article, it is shown that HPLC techniques, given their wide versatility, relative ease of use, and high resolution, may be considered the most valuable tool for the characterization of virtually any hydrophobic protein. Application examples are described, and comparisons with other methods are discussed. Moreover, HPLC is not a destructive technique, and therefore, proteins, once separated, are available for further analytical investigations. Among these techniques, quantitative and qualitative analyses of the separated fractions can be obtained through other biophysical approaches, such as crystallography or structural spectroscopy. Most of these approaches require preliminary protein purification (90% or higher), which could be rapidly obtained through preliminary HPLC.

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Purification of human serum amyloid A by anion-exchange fast protein liquid chromatography

Cited by 11 publications

References 21 publications

Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum – a pilot study

Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum – a pilot study

Chromatography of Membrane Proteins and Lipoproteins

Chromatography of Membrane Proteins and Lipoproteins

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