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Anal Biochem. 1996 Nov 1;242(1):26-30.

The visualization of peroxisomal proteins containing a C-terminal targeting sequence on western blot by using the biotinylated PTS1-receptor.

Author information

1
Departement Moleculaire Celbiologie, Katholieke Universiteit Leuven-Fakulteit Geneeskunde, Belgium.

Erratum in

  • Anal Biochem 1997 Mar 15;246(2):270.

Abstract

A procedure to visualize proteins containing a C-terminal peroxisomal targeting signal (PTS1) in complex protein mixtures was developed using a bacterially expressed, biotinylated form of the human PTS1-receptor. The binding of this fusion product to purified PTS1-containing proteins that were separated by SDS-PAGE and blotted onto nitrocellulose was detected by means of streptavidin-alkaline phosphatase and shown to be both saturable and specific. When applied to total tissue extracts, in addition to PTS1-containing proteins various endogenous biotinylated proteins were visualized. Therefore, a two-step staining procedure was optimized whereby the endogenous biotinylated proteins were shielded with a blue precipitate, followed by incubation with the biotinylated receptor and detection of the resulting PTS1-receptor/PTS1-protein complexes with a phosphatase reaction coupled to the formation of a red-colored precipitate. This relatively inexpensive, simple, and fast technique enabled us to visualize a variety of PTS1-containing proteins. In addition, the information presented in this study can be used to facilitate the identification and characterization of receptor-ligand interaction in general and to eliminate interference by endogenous biotinylated proteins intrinsic to the streptavidin-biotin detection system.

PMID:
8923960
DOI:
10.1006/abio.1996.0423
[Indexed for MEDLINE]

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