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.

Guided by the source plate map, load 1 to 2 microliters of each peptide feature into the designated wells of one or more 384-well small-volume plates. Dilute each feature tenfold with 1x protein microarray printing buffer, supplemented with 1% bovine serum albumin, and 5 micrograms per microliter fluorescein-labeled biotin. Then, centrifuge the plates at 500 times g at room temperature for 2 minutes. Next, empty the waste container of the microarray printer. Fill the wash solution in humidifier containers with sterile distilled water. Enter the array slide parameters in the microarray printer software.

Set the washing procedure to a 1-second wash with a single immersion, the post-wash to redip the pins five times, and the humidity to 60%. Then, insert streptavidin-coated glass slides into the substrate platens. Load the platens into the platen elevator. Insert the source plates into the plate holders, and load the holders into the source plate elevator. Run the printing process.

Then, remove the substrate platens from the instrument. Block the printed slides with 1x protein microarray blocking buffer for 30 minutes while shaking. Then, wash the slides in room-temperature pH 7.6 phosphate-buffered saline for 10 minutes. Repeat the wash with a fresh batch of PBS. Dry the slides by centrifugation for 30 seconds at room temperature.

Next, preheat a microarray wax imprinter at 85 degrees Celsius for 30 minutes, or until the wax has melted. Then, insert a slide, printed side down, into the holder, and align the holder with the imprinter mold. Bring the mold into contact with the slide, and hold for two seconds. Then, quickly remove the slide from the holder and inspect the wax borders to verify that the arrays are properly enclosed. Store the partitioned slides at 4 degrees Celsius in a dry, dark area.

To begin the hybridization procedure, place a partitioned array slide in a plastic dish, and cover the slide with hybridization buffer. Equilibrate the slide for 30 minutes at 4 degrees Celsius while shaking at low speed. Dry the slide by spinning in a microarray slide centrifuge at room temperature. Then, add 5 microliters of each histone PTM antibody solution to at least two wells of the array, and incubate at 4 degrees Celsius for one hour.

Following incubation, remove the antibody solution, and wash the array with cold PBS three times, for 5 minutes each, at 4 degrees Celsius. Next, incubate the array in a fluorescent dye conjugated secondary antibody solution for 30 minutes at 4 degrees Celsius, in the absence of light. Wash the slide three times with cold PBS as before.

Dip the array in room temperature 0.1x PBS to remove excess salts, and dry the slide by brief centrifugation at room temperature. Image the slide with a microarray scanner at a resolution of at least 25 microns.