Encyclopedia of Experiments: Immunology
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Transcript
Take a microarray slide containing histone peptides with target and non-target post-translational modifications or PTMs.
The peptides are immobilized via conjugated biotin at specific spots on the streptavidin-functionalized glass slide.
The spots also contain an immobilized biotin-conjugated green fluorophore, facilitating the identification of the spots.
Introduce a blocking buffer, preventing non-specific antibody interaction.
Remove the buffer and centrifuge to dry the slide.
Inside a wax imprinter, border the array with wax.
Equilibrate the array with a hybridization buffer.
Incubate with the primary antibodies specific for a target PTM.
Introduce red fluorophore-conjugated secondary antibodies that bind to the primary antibodies.
Remove unbound antibodies and centrifuge to dry the slide.
Image the slide using a microarray scanner. The green fluorescence helps identify the spots, while the red fluorescence indicates PTM-antibody interaction, demonstrating antibody specificity.
Recognition of the target PTM demonstrates antibody specificity, whereas recognition of non-target PTMs indicates antibody cross-reactivity.
