Non-immunological detection of PhoA fusion proteins

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

doi:10.1016/0167-7012(92)90020-5

Non-immunological detection of PhoA fusion proteins

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

Research output: Contribution to journal › Article › peer-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 language	English (US)
Pages (from-to)	13-22
Number of pages	10
Journal	Journal of Microbiological Methods
Volume	16
Issue number	1
DOIs	https://doi.org/10.1016/0167-7012(92)90020-5
State	Published - Aug 1992
Externally published	Yes

Keywords

Alkaline phosphatase
Fusion proteins
Western blot

ASJC Scopus subject areas

Microbiology
Molecular Biology
Microbiology (medical)

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10.1016/0167-7012(92)90020-5

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title = "Non-immunological detection of PhoA fusion proteins",

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.",

keywords = "Alkaline phosphatase, Fusion proteins, Western blot",

author = "Phillips, \{Linda H.\} and Chambers, \{La Shauna\} and Ricardo Belmares and Niesel, \{David W.\}",

year = "1992",

month = aug,

doi = "10.1016/0167-7012(92)90020-5",

language = "English (US)",

volume = "16",

pages = "13--22",

journal = "Journal of Microbiological Methods",

issn = "0167-7012",

publisher = "Elsevier B.V.",

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}

TY - JOUR

T1 - Non-immunological detection of PhoA fusion proteins

AU - Phillips, Linda H.

AU - Chambers, La Shauna

AU - Belmares, Ricardo

AU - Niesel, David W.

PY - 1992/8

Y1 - 1992/8

N2 - 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.

AB - 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.

KW - Alkaline phosphatase

KW - Fusion proteins

KW - Western blot

UR - https://www.scopus.com/pages/publications/0026638896

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U2 - 10.1016/0167-7012(92)90020-5

DO - 10.1016/0167-7012(92)90020-5

M3 - Article

AN - SCOPUS:0026638896

SN - 0167-7012

VL - 16

SP - 13

EP - 22

JO - Journal of Microbiological Methods

JF - Journal of Microbiological Methods

IS - 1

ER -

Non-immunological detection of PhoA fusion proteins

Abstract

Keywords

ASJC Scopus subject areas

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