Non-immunological detection of PhoA fusion proteins

Linda H. Phillips, La Shauna Chambers, Ricardo Belmares, David W. Niesel

    Research output: Contribution to journalArticlepeer-review

    3 Scopus citations

    Abstract

    A non-immunological method for the detection of PhoA fusion proteins on Western blots has been developed. Bands representing the alkaline phosphatase-active species of the fusion proteins are visualized after incubation with the colorimetric alkaline phosphatase substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Blocking the filters in 3% gelatin in Tris buffered saline containing 0.05% Tween-20 prior to color development is essential, and reactions are enhanced by extending blocking and development times. Fusion proteins can be detected over a broad protein concentration range. This method is based upon the ability of the fusion proteins to renature and regain enzymatic activity after immobilization on nitrocellulose.

    Original languageEnglish (US)
    Pages (from-to)13-22
    Number of pages10
    JournalJournal of Microbiological Methods
    Volume16
    Issue number1
    DOIs
    StatePublished - Aug 1992

    Keywords

    • Alkaline phosphatase
    • Fusion proteins
    • Western blot

    ASJC Scopus subject areas

    • Microbiology
    • Molecular Biology
    • Microbiology (medical)

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