Non-immunological detection of PhoA fusion proteins

Linda H. Phillips, LaShauna Chambers, Ricardo Belmares, David Niesel

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 - 1992

Fingerprint

Proteins
Alkaline Phosphatase
Nitroblue Tetrazolium
Collodion
Polysorbates
Gelatin
Immobilization
Color
Western Blotting
5-bromo-4-chloro-3-indoxyl phosphate

Keywords

  • Alkaline phosphatase
  • Fusion proteins
  • Western blot

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

Non-immunological detection of PhoA fusion proteins. / Phillips, Linda H.; Chambers, LaShauna; Belmares, Ricardo; Niesel, David.

In: Journal of Microbiological Methods, Vol. 16, No. 1, 1992, p. 13-22.

Research output: Contribution to journalArticle

Phillips, Linda H. ; Chambers, LaShauna ; Belmares, Ricardo ; Niesel, David. / Non-immunological detection of PhoA fusion proteins. In: Journal of Microbiological Methods. 1992 ; Vol. 16, No. 1. pp. 13-22.
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