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    Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis


    Mulvey, Claire and Ohlendieck, Kay (2003) Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis. Analytical Biochemistry, 319 (1). pp. 122-130. ISSN 0003-2697

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    Abstract

    Blot overlay techniques have long been used to directly visualize protein-protein interactions within membrane complexes. However, this approach is often hampered by the limited quantities of purified membrane proteins available for conjugation with marker molecules. Here we applied continuous-elution gel electrophoresis as a preparative alternative to isolate sufficient amounts of a homogeneous protein sample to be used as a peroxidase-labeled probe in blot overlays. Microsomal muscle proteins ranging from approximately 20 to 600 kDa were electrophoretically separated and various marker proteins present in eluted fractions were identified by immunoblotting. Since the supramolecular structure of calsequestrin has recently been determined, this terminal cisternae protein was isolated as a model protein for studying protein-protein interactions. In blot overlay assays, peroxidase-conjugated calsequestrin specifically bound to the ryanodine receptor, triadin, calsequestrin itself, and junctin, illustrating that the biological binding affinities are retained in electrophoretically prepared muscle proteins. Potential applications for differential blot overlay approaches and for analyzing pathophysiological preparations from dystrophic muscle were evaluated. Since continuous-elution gel electrophoresis can separate a wide range of differently sized proteins from subcellular fractions, our report indicates that this technique can be utilized for the rapid identification of protein-protein interactions in future high-throughput analyses of subproteomes.

    Item Type: Article
    Keywords: Blot overlay; Calsequestrin; Continuous-elution gel electrophoresis; Protein oligomerization; Supramolecular complex;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 7514
    Identification Number: https://doi.org/10.1016/S0003-2697(03)00321-X
    Depositing User: Prof. Kay Ohlendieck
    Date Deposited: 14 Oct 2016 14:51
    Journal or Publication Title: Analytical Biochemistry
    Publisher: Elsevier Masson
    Refereed: Yes
    Funders: Enterprise Ireland (EI), Health Research Board (HRB), Muscular Dystrophy Ireland, European Commission
    URI:

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