Reverse Phase Protein Arrays Based Protein Expression Analysis: A Procedure for Simultaneous Quantification of Expression of Multiple Proteins from Cell Lysate

Prior to RPPA printing, a series of five twofold dilutions are made from each sample. One of the most time-consuming aspects of this procedure is making hundreds of dilutions. Successful outcome can be assured by double-checking prior to printing slides.

A MicroGrid II robotics spotter is then used to spot protein lysates onto nitrocellulose-coated glass slides. Each sample is spotted in triplicate, resulting in a total of 15 spots per sample. The slides used in this experiment contain two pads onto which the samples are spotted. Each pad was spotted with identical samples, in this case, 100 samples. Other available formats include 1, 8, and 16 pad slides. The higher the number of pads, the smaller the number of samples that can be spotted onto each.

To begin the procedure for protein detection, wet slides in excess blocking buffer and incubate at room temperature for 1 hour on a rocking platform. Prepare 800 microliters of primary antibodies in blocking buffer at the desired concentrations and keep on ice. After the one-hour incubation in blocking buffer, mount slides in either the single frame Chip Clip or the FastFrame four-bay slide holder so that a tight seal is formed between the slide and the incubation chamber.

Remove residual buffer from wells and add 600 microliters of primary antibody to the respective wells. Place the slides and chamber into a sealed wet box and incubate on a rocking platform overnight at 4 degrees Celsius. On the following morning, remove slides from the cold room. Carefully remove the primary antibodies from each well. Add 600 microliters of 0.1% PBS-Tween20 or PBS-T and wash slides on a rocking platform at room temperature for 5 minutes.

Replace the PBS-T with fresh PBS-T. In this way, wash slides a total of three times. Remove buffer from wells and add 600 microliters of fluorescently-labeled secondary antibodies at the appropriate dilution to the respective wells. Incubate with secondary antibodies at room temperature for 45 minutes with gentle shaking. It is important to protect the membrane from light until the time it is scanned. After 45 minutes, remove secondary antibodies from wells and briefly washed three times in 600 microliters PBS-T at room temperature.

Remove slides from the carrier, transfer to a suitable container, and wash in excess PBS-T for 15 minutes, keeping the membrane in the dark. Remove PBS-T and wash the membrane with PBS at room temperature for 15 minutes to remove residual Tween20, again keeping the membrane in the dark. Dry the Fastslides in a 50 degrees Celsius oven for 10 minutes. Finally, scan the slides at 680 nanometers and/or 800 nanometers, depending on the secondary antibodies used.