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Optimization of a malachite green assay for detection of ATP hydrolysis by solubilized membrane proteins.

Abstract

We studied the activity of the fluorescently labeled membrane transporter MalGFK(2), which transports maltose at the expense of ATP hydrolysis. We used a commercially available malachite green assay (SensoLyte MG phosphate assay kit; Anaspec) to quantify the liberated phosphate upon ATP hydrolysis. However, strong variations in phosphate concentration were measured when using the supplier's handling protocol. We optimized the protocol, taking into account the effects mediated by glycerol, SDS, and fluorescent label on the sample. As a result we obtained highly reproducible phosphate concentration values under conditions optimal for solubilized membrane proteins.

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  • Authors+Show Affiliations

    ,

    Institut für Experimentalphysik, Freie Universität Berlin, D-14195 Berlin, Germany.

    ,

    Source

    Analytical biochemistry 426:2 2012 Jul 15 pg 103-5

    MeSH

    Adenosine Triphosphate
    Biological Products
    Glycerol
    Hydrolysis
    Membrane Proteins
    Phosphates
    Rosaniline Dyes
    Sodium Dodecyl Sulfate

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    22507377

    Citation

    * When formatting your citation, note that all book, journal, and database titles should be italicized* Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Optimization of a malachite green assay for detection of ATP hydrolysis by solubilized membrane proteins. AU - Repen,Boris, AU - Schneider,Erwin, AU - Alexiev,Ulrike, Y1 - 2012/04/13/ PY - 2012/01/16/received PY - 2012/04/08/revised PY - 2012/04/10/accepted PY - 2012/4/18/entrez PY - 2012/4/18/pubmed PY - 2012/10/2/medline SP - 103 EP - 5 JF - Analytical biochemistry JO - Anal. Biochem. VL - 426 IS - 2 N2 - We studied the activity of the fluorescently labeled membrane transporter MalGFK(2), which transports maltose at the expense of ATP hydrolysis. We used a commercially available malachite green assay (SensoLyte MG phosphate assay kit; Anaspec) to quantify the liberated phosphate upon ATP hydrolysis. However, strong variations in phosphate concentration were measured when using the supplier's handling protocol. We optimized the protocol, taking into account the effects mediated by glycerol, SDS, and fluorescent label on the sample. As a result we obtained highly reproducible phosphate concentration values under conditions optimal for solubilized membrane proteins. SN - 1096-0309 UR - https://www.unboundmedicine.com/medline/citation/22507377/Optimization_of_a_malachite_green_assay_for_detection_of_ATP_hydrolysis_by_solubilized_membrane_proteins_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(12)00228-X DB - PRIME DP - Unbound Medicine ER -