Reverse-phase Protein Arrays
The Reverse Phase Protein Array (RPPA) is a flexible, high density protein lysate microarray that offers key advantages over alternative proteomic technologies through its sensitivity, sample throughput, data consistency and speed.
Sensitivity: Low Input Amounts of Protein.
The RPPA platform can be several orders of magnitude more sensitive than technologies such as Western blots or ELISA. As an example, the amount of protein that is typically loaded into one lane for Western blot analysis is enough to perform hundreds to thousands of assays.
- A key aspect of this low protein requirement is that it allows for the comprehensive mapping of druggable targets and their downstream pathways from 48 or 96 well cell cultures, depending upon the cell type and percent confluency.
- This ability to work out of small-sized cell cultures is important for the early stages of evaluating a compound (or compounds in a series) when the amount of the test articles available may be limited.
- Furthermore, by downsizing the size of cell culture needed for proteomic analysis, media and labor cost can be reduced, as well as the time needed to grow up and seed larger cell culture plates (a typical Western blot study utilizes 6-well culture plates).
- For studies involving tissue samples (e.g. xenograft tissue blocks, core needle biopsies, etc.), the same ability to analyze large numbers of targets and pathways from small amounts of starting material also is often available from single tissue sections. In contrast, immunohistochemistry (IHC) analysis requires at least one tissue section (and more commonly 2 to 3 sections) to analyze a single protein marker.
- The proteomic analysis capabilities of the RPPA platform also extend to other small sample preparations such as peripheral blood mononuclear cells (PBMCs), immune-separated blood cell populations, micropunches, laser capture microdissected cell populations, etc.
Sample Throughput and Consistency – Effect on Study Design.
The ability to multiplex hundreds of samples simultaneously when using the RPPA platform offers the opportunity to achieve greater throughput than technologies such as Western blots (which are typically limited to experiments that can easily fit within the standard 12 or 20 well lanes) or Mass Spec (tens of samples per week).
- Unlike Western blots, hundreds of samples can be processed simultaneously in the presence of positive controls and calibration standards (the latter can be used to help compare results between different studies performed over time).
- Furthermore, quantitative and statistical data are obtainable from each sample in a format that allows for bioinformatic analysis to be performed in a similar manner to the data obtained from standard gene expression microarrays.
- In situ protein quantification is performed for each printed protein lysate spot that is used to help normalize or account for technical variability that may occur between printed samples.
Overall, the capacity to analyze up to 300 samples simultaneously allows for greater experimental flexibility, and enables researchers the ability to examine more numerous variables such as cell types, dose response, time course, treatment arms with pr-and post-dose, etc, than is normally done in alternative technologies such as Western blot or Mass Spec analysis.
When supplied with protein lysates, hundreds of samples can be analyzed using tens to hundreds of RPPA assays with a turn-around time typically ranging from 5 to 15 business days.
For further information on Theranostics Health’s proteomic capabilities, currently available assays and a list of peer-reviewed publications, please select one of the following:
For a listing of currently available RPPA assays, please select Available RPPA Assays – June 2016
For further information, please contact Theranostics Health directly with your inquiries.