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

Published: April 30, 2023

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 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. A series of five 2-fold dilutions were made fro…

Representative Results

Figure 1. Flow diagram of RPPA scanning process. 

開示

The authors have nothing to disclose.

Materials

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

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記事を引用
Reverse Phase Protein Arrays Based Protein Expression Analysis: A Procedure for Simultaneous Quantification of Expression of Multiple Proteins from Cell Lysate. J. Vis. Exp. (Pending Publication), e20571, doi: (2023).

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