Validation of the biotinyl ligand-avidin-gold technique
| Autor: | Randal E. Morris, Catharine B. Saelinger, Georgianne M. Ciraolo |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Streptavidin
Cholera Toxin Histology Virulence Factors Wheat Germ Agglutinins Endosome media_common.quotation_subject Bacterial Toxins Biotin Exotoxins Golgi Apparatus Biology Cell Line Mice chemistry.chemical_compound symbols.namesake Agglutinin Animals Internalization media_common ADP Ribose Transferases Fibroblasts Golgi apparatus Avidin Immunohistochemistry Endocytosis chemistry Biochemistry Biotinylation symbols Biophysics biology.protein Anatomy Lysosomes Intracellular |
| Zdroj: | Journal of Histochemistry & Cytochemistry. 40:711-721 |
| ISSN: | 1551-5044 0022-1554 |
| DOI: | 10.1177/40.5.1573251 |
| Popis: | We demonstrate here that the intracellular routing of biotinylated ligands was not affected by the attachment of streptavidin gold colloids so long as the electron-dense marker was added after the biotinyl ligand-receptor interaction had occurred. The binding, internalization, and intracellular routing of three different biotinyl ligands were followed in mouse LM fibroblasts. The biotinyl (B) ligands included B-choleragenoid (B-CTd), B-wheat germ agglutinin (B-WGA), and B-Pseudomonas exotoxin A (B-PE). All three ligands showed distinct intracellular trafficking patterns. B-WGA and B-PE entered via clathrin-coated pits, whereas B-CTd did not. After entry, B-CTd was routed to the lysosomal compartment without involvement of the Golgi. Although B-PE and B-WGA were also routed to the lysosomal compartment, a significant portion of these two ligands was observed in association with the Golgi. B-WGA, however, remained in the endosomal and Golgi compartments longer than did B-PE. We also monitored the internalization and routing of native PE by an indirect immunoperoxidase technique done in conjunction with saponin solubilization. The results corroborated the observations with the biotinyl-PE-streptavidin-gold method. In contrast, biotinyl-PE added to streptavidin-gold before addition to LM cells was poorly internalized and routed aberrantly. From these observations we conclude that the biotinyl ligand-avidin-gold technique is a valid method for following the binding, internalization, and intracellular routing of ligands. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|