Identification of microinjected cells using biotinylated antibodies and Strep-avidin-conjugated horseradish peroxidase.
Results from experiments using needle microinjection of cells are often compromised by an inability to readily demonstrate which cells within a population have been injected. The technique described here allows the unambiguous identification of cells that have been successfully microinjected. Sequential incubation of fixed cells with biotinylated anti-immunoglobulin antibodies, followed by horseradish peroxidase (HRP)-conjugated Strep-avidin and HRP substrate, provides a sensitive assay for identification of cells containing trace amounts of immunoglobulins. This allows direct correlation to the presence of injected molecules of effects on cell morphology, the ability to enter into DNA synthesis, or expression of specific genes. By a variety of criteria, nonspecific immunoglobulins do not adversely affect cellular processes when injected by themselves or in the presence of other proteins known to have biological effects when injected, such as cAMP-dependent protein kinase and the ras oncogene protein.
Riabowol
K T
KT
Cold Spring Harbor Laboratory, New York 11724.
D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D001426:Bacterial Proteins; D001710:Biotin / Q000378:metabolism*; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology*; D006735:Horseradish Peroxidase; D008845:Microinjections; D010544:Peroxidases
D000906:Antibodies; D001426:Bacterial Proteins; C058865:Strep-avidin conjugated horseradish peroxidase; D001710:Biotin; D006735:Horseradish Peroxidase; D010544:Peroxidases