| Quantitative analysis of immobilized proteins and protein mixtures by amino acid analysis. | |
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MedLine Citation:
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PMID: 12924786 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Biomolecular surface engineering of materials often requires precise, versatile and efficient quantification of immobilized proteins at solid surfaces. Acidic hydrolysis of surface-bound proteins and subsequent HPLC analysis of fluorescence-derivatized amino acids were adapted and critically evaluated for that purpose. Contaminations and concentration-dependent amino acid retrieval during HPLC were found to influence the accuracy of the method. In addition to the choice of adequate conditions for hydrolysis, derivatization and chromatographic separation extensions of the data evaluation were suggested to improve the accuracy of the approach when applied to single protein systems: comparing the experimentally obtained amino acid ratio to the protein constitution enabled to identify the properly separated and detected amino acids. Those amino acids were selected for a more precise calculation of the amount of immobilized protein. To further increase the accuracy of the method, the retrieval of amino acids corresponding to protein amounts in the range between 0.5 and 4.0 microg was analyzed for a variety of proteins of interest to derive protein-specific correction factors. The evaluation of amino acid data was furthermore applied to quantify binary protein mixtures at similar settings. This method was proven useful to detect the composition of protein mixtures throughout a wide range of absolute and relative concentrations. |
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Authors:
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Katrin Salchert; Tilo Pompe; Claudia Sperling; Carsten Werner |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Journal of chromatography. A Volume: 1005 ISSN: 0021-9673 ISO Abbreviation: J Chromatogr A Publication Date: 2003 Jul |
Date Detail:
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Created Date: 2003-08-19 Completed Date: 2004-03-24 Revised Date: 2009-01-15 |
Medline Journal Info:
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Nlm Unique ID: 9318488 Medline TA: J Chromatogr A Country: Netherlands |
Other Details:
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Languages: eng Pagination: 113-22 Citation Subset: IM |
Affiliation:
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Institut für Polymerforschung Dresden e.V. Abteilung Biokompatible Materialien, Hohe Str. 6, 01069 Dresden, Germany. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acids
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analysis* Chromatography, High Pressure Liquid / methods Fluorescent Dyes / chemistry Proteins / analysis*, chemistry Spectrometry, Fluorescence |
| Chemical | |
Reg. No./Substance:
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0/Amino Acids; 0/Fluorescent Dyes; 0/Proteins |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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