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

Abstract

Source: O'Mahony, F. C. et al. The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers. J. Vis. Exp. (2013)

This video demonstrates the methodology for performing Reverse Phase Protein Arrays (RPPA) to study protein expression patterns from cell lysate. In RPPA, the lysate containing a mixture of proteins is printed on nitrocellulose slides and proteins of interest are analyzed using fluorescently labeled antibodies.

Protocol

1. RPPA Printing

1. Protein lysates were spotted onto nitrocellulose-coated glass slides (Fastslides-Whatman) using a MicroGrid II robotic spotter. The slides used contained 2 pads onto which the samples were 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 which can be spotted onto each.
2. A series of five 2-fold dilutions were made from each sample and each was then spotted in triplicate (resulting in a total of 15 spots per sample).

2. RPPA ....

Results

Figure 1. Flow diagram of RPPA scanning process.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Triton X-100	Triton-X	T8787
Li-Cor Odyssey Blocking Buffer	Li-Cor	927-40000
MicroGrid II robotic spotter	Biorobotics
FastFrame' four bay slide holder	Whatman	10486001
FAST Slide - 2-Pad	Whatman	10485317
IRDye 680LT Goat anti-Mouse IgG	Licor	926-68020
IRDye 800CW Goat anti-Rabbit IgG	Licor	926-32211