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.
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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 |
