English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Production and characterization of monoclonal antibodies directed against native epitopes of NhaA, the Na+/H+ antiporter of Escherichia coli

Padan, E., Venturi, M., Michel, H., & Hunte, C. (1998). Production and characterization of monoclonal antibodies directed against native epitopes of NhaA, the Na+/H+ antiporter of Escherichia coli. FEBS Letters, 441(1), 53-58. doi:10.1016/s0014-5793(98)01524-5.

Item is

Basic

show hide
Genre: Journal Article

Files

show Files

Locators

show

Creators

show
hide
 Creators:
Padan, Etana1, Author
Venturi, Miro2, Author           
Michel, Hartmut2, Author           
Hunte, Carola2, Author           
Affiliations:
1Division of Microbial and Molecular Ecology, The Hebrew University of Jerusalem, Israel, ou_persistent22              
2Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

Content

show
hide
Free keywords: Active transport; Na+/H+ antiporter; NhaA; Monoclonal antibody; Escherichia coli
 Abstract: Monoclonal antibodies (mAbs) recognizing native epitopes are an important tool for functional and structural studies of proteins, yet they have rarely been used with transport proteins. In an attempt to raise monoclonal antibodies against the NhaA Na+/H+ antiporter of Escherichia coli we encountered difficulties in the screening procedure, which is based on the standard enzyme-linked immunosorbent assay (ELISA). Here we report a rapid and efficient method of screening for anti-NhaA mAbs which recognize native epitopes of the antiporter. The method is based on the use of His-tagged protein, Ni2+-nitrilotriacetic acid coated plates and non-denaturing conditions in the assay. With this procedure four mAbs were obtained, three of which recognize the NhaA in its native conformation and one preferentially recognizes the denatured form. The latter mAb is Western blot positive, the others are Western blot negative and bind the detergent solubilized NhaA as assayed by gel filtration chromatography. Competition experiments show that the native epitopes are common to both the His-tagged and the wild-type protein. We suggest that in the standard ELISA the NhaA protein is not presented to the antibody in the native conformation whereas the His tag based protocol favors this presentation. Indeed, we could remarkably improve the low reactivity of the standard ELISA by coating the plates with anti-NhaA mAb and use it to present NhaA ('sandwich' ELISA or two antibodies assay). Remarkably, two of the mAbs (5H4, 2C5) which bind native NhaA inhibit drastically the deltapH driven 22Na uptake mediated by His-tagged NhaA reconstituted in proteoliposomes. Hence, these mAbs afford a new tool to study the structure/function relationship of the antiporter.

Details

show
hide
Language(s): eng - English
 Dates: 1998-11-061998-12-301998-12-11
 Publication Status: Published in print
 Pages: 6
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1016/s0014-5793(98)01524-5
PMID: 9877164
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: FEBS Letters
  Other : FEBS Lett.
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 441 (1) Sequence Number: - Start / End Page: 53 - 58 Identifier: ISSN: 0014-5793
CoNE: https://pure.mpg.de/cone/journals/resource/954925399501