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      Development of a Univariate Membrane-Based Mid-Infrared Method for Protein Quantitation and Total Lipid Content Analysis of Biological Samples

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          Abstract

          Biological samples present a range of complexities from homogeneous purified protein to multicomponent mixtures. Accurate qualification of such samples is paramount to downstream applications. We describe the development of an MIR spectroscopy-based analytical method offering simultaneous protein quantitation (0.25–5 mg/mL) and analysis of total lipid or detergent species, as well as the identification of other biomolecules present in biological samples. The method utilizes a hydrophilic PTFE membrane engineered for presentation of aqueous samples in a dried format compatible with fast infrared analysis. Unlike classical quantification techniques, the reported method is amino acid sequence independent and thus applicable to complex samples of unknown composition. By comparison to existing platforms, this MIR-based method enables direct quantification using minimal sample volume (2  µL); it is well-suited where repeat access and limited sample size are critical parameters. Further, accurate results can be derived without specialized training or knowledge of IR spectroscopy. Overall, the simplified application and analysis system provides a more cost-effective alternative to high-throughput IR systems for research laboratories with minimal throughput demands. In summary, the MIR-based system provides a viable alternative to current protein quantitation methods; it also uniquely offers simultaneous qualification of other components, notably lipids and detergents.

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          Most cited references48

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          Activities at the Universal Protein Resource (UniProt)

          The mission of the Universal Protein Resource (UniProt) (http://www.uniprot.org) is to provide the scientific community with a comprehensive, high-quality and freely accessible resource of protein sequences and functional annotation. It integrates, interprets and standardizes data from literature and numerous resources to achieve the most comprehensive catalog possible of protein information. The central activities are the biocuration of the UniProt Knowledgebase and the dissemination of these data through our Web site and web services. UniProt is produced by the UniProt Consortium, which consists of groups from the European Bioinformatics Institute (EBI), the SIB Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR). UniProt is updated and distributed every 4 weeks and can be accessed online for searches or downloads.
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            Capillary flow as the cause of ring stains from dried liquid drops

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              Fourier transform infrared spectroscopic analysis of protein secondary structures.

              Infrared spectroscopy is one of the oldest and well established experimental techniques for the analysis of secondary structure of polypeptides and proteins. It is convenient, non-destructive, requires less sample preparation, and can be used under a wide variety of conditions. This review introduces the recent developments in Fourier transform infrared (FTIR) spectroscopy technique and its applications to protein structural studies. The experimental skills, data analysis, and correlations between the FTIR spectroscopic bands and protein secondary structure components are discussed. The applications of FTIR to the secondary structure analysis, conformational changes, structural dynamics and stability studies of proteins are also discussed.
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                Author and article information

                Journal
                J Anal Methods Chem
                J Anal Methods Chem
                JAMC
                Journal of Analytical Methods in Chemistry
                Hindawi Publishing Corporation
                2090-8865
                2090-8873
                2014
                13 October 2014
                : 2014
                : 657079
                Affiliations
                1EMD Millipore Corporation, 17 Cherry Hill Drive, Danvers, MA 01923, USA
                2Institute for Organic Chemistry and Biochemistry, Technical University Darmstadt, 64289 Darmstadt, Germany
                3Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany
                Author notes

                Academic Editor: Bernd Hitzmann

                Author information
                http://orcid.org/0000-0003-0010-7986
                http://orcid.org/0000-0002-3760-4735
                http://orcid.org/0000-0002-8343-4909
                http://orcid.org/0000-0002-0052-0554
                http://orcid.org/0000-0003-4082-2386
                http://orcid.org/0000-0002-3567-8765
                http://orcid.org/0000-0002-3898-1743
                Article
                10.1155/2014/657079
                4211209
                9657e5bd-6bc3-4f67-9562-ee72b27db147
                Copyright © 2014 Ivona Strug et al.

                This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 1 July 2014
                : 1 September 2014
                Categories
                Research Article

                Analytical chemistry
                Analytical chemistry

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