The visualization of peroxisomal proteins containing a C-terminal targeting sequence on western blot by using the biotinylated PTS1-receptor.
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.
Fransen
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M
Departement Moleculaire Celbiologie, Katholieke Universiteit Leuven-Fakulteit Geneeskunde, Belgium.
Brees
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Van Veldhoven
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Mannaerts
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D000469:Alkaline Phosphatase; D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin / Q000031:analogs & derivatives*; D015153:Blotting, Western / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008830:Microbodies / Q000737:chemistry*; D000074435:Peroxisome-Targeting Signal 1 Receptor; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D019809:Streptavidin; D013347:Subcellular Fractions / Q000737:chemistry
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D000469:Alkaline Phosphatase