Labeling and purification of cellulose-binding proteins for high resolution fluorescence applications

Jose M. Moran-Mirabal, Stephane C. Corgie, Jacob C. Bolewski, Hanna M. Smith, Benjamin R. Cipriany, Harold G. Craighead, Larry P. Walker

    Research output: Contribution to journalArticlepeer-review

    14 Scopus citations

    Abstract

    The study of enzymatic reactions through fluorescence spectroscopy requires the use of bright, functional fluorescent molecules. In the case of proteins, labeling with fluorescent dyes has been carried out through covalent reactions with specific amino acids. However, these reactions are probabilistic and can yield mixtures of unlabeled and labeled enzymes with catalytic activities that can be modified by the addition of fluorophores. To have meaningful interpretations of results from the study of labeled enzymes, it is then necessary to reduce the variability in physical, chemical, and biological characteristics of the labeled products. In this paper, a solid phase labeling protocol is described as an advantageous alternative to free solution labeling of cellulose-binding proteins and is applied to tag cellulases with three different fluorophores. The products from the labeling reactions were purified to remove the unreacted dye and separate labeled and unlabeled enzymes. Characterization of the catalytic and spectroscopic properties of the isolated labeled species confirmed that highly homogeneous populations of labeled cellulases can be achieved. The protocol for the separation of labeled products is applicable to any mixture of labeled proteins, making this an attractive methodology for the production of labeled proteins suitable for single molecule fluorescence spectroscopy.

    Original languageEnglish (US)
    Pages (from-to)7981-7987
    Number of pages7
    JournalAnalytical Chemistry
    Volume81
    Issue number19
    DOIs
    StatePublished - Oct 1 2009

    ASJC Scopus subject areas

    • Analytical Chemistry

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