Detection of Vitronectin Binding to Bacterial Surfaces using Flow Cytometry
Transcrição
Take flow cytometer tubes containing wild-type and mutant strains of Haemophilus influenzae, type f.
Pipette a buffer containing blocking proteins and vitronectin. Incubate.
The blocking proteins reduce non-specific interactions on the bacterial surface, while vitronectin molecules bind to protein H, a vitronectin-binding protein, on wild-type bacterial surfaces. In the mutant strain, vitronectin does not bind due to the absence of protein H.
Centrifuge. Remove unbound vitronectin and blocking proteins.
Add a buffer containing primary anti-vitronectin antibodies, which specifically bind to vitronectin attached to the wild-type bacterial surfaces.
Next, introduce fluorophore-conjugated secondary antibodies, which bind to the primary antibodies on vitronectin and facilitate vitronectin detection.
Centrifuge. Remove unbound secondary antibodies. Resuspend bacteria in the buffer.
Perform flow cytometry to determine vitronectin binding to the bacterial surface.
Compare the fluorescence signals of wild-type and mutant strains. A shift in the fluorescence signal in wild-type bacteria confirms vitronectin binding to the bacterial surface.
