A Peptide Microarray Technology for Specificity Profiling of Antibodies

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.