Reverse phase protein array technology is a high-throughput antibody-based technique that allows for rapid exploratory proteomics on small sample populations.
RPPA technology enables researchers to perform what would ordinarily be an extremely large number of western blots in a highly sophisticated and very high throughput manner. Proteins are spotted onto specialized glass slides, bound and blocked, processed with appropriate primary and secondary antibodies then quantitated using a proprietary reader that uses specialised technology to quantitate accurate fluorescence levels. The system requires only very small amounts of protein samples; from standard cell-culture sourced material and complex 3D cultures through to freshly extracted patient samples. Large numbers of analytes can be analysed and quantitated very rapidly.
The RPPA Platform, housed at the Peter MacCallum Cancer Centre, is a dedicated service facility open to researchers on a fee-for-service basis with the fundamental core value of enabling researchers to perform the most sophisticated experiments. The platform offers experimental design guidance, direct lysate submission, selection of a vast range of verified antibodies for your screen and bioinformatic analysis. Researchers can then validate their experimental outcomes using traditional western blotting with the same reagents.
In a nutshell, protein lysates are spotted into specialised glass slides in grid patterns that enable us to spot 66 proteins per grid box, each grid box is one antibody. You can analyse more samples than 66, it just means we spot into a second grid box. If screening one glass slide, you can screen 4 antibodies and 2 controls. Subsequent slides do not need any controls and therefore you can screen 6 antibodies per slide. These permutations are explained as an overview in the pricing guide and in detail in the user guide.
The RPPA platform comprises a GeSim Nanoplotter for spotting protein samples and a Zeptosens ZeptoReader, a laser based scanning and software system to image and quantify expression. All westerns are quantified using IR-labelled secondary antibody detection on a LiCOR Odyssey scanner. We use large scale liquid handling automation (ALH3000 Sciclone, Perkin Elmer) for all protein lysate dilutions.
Researchers have accessed this platform to perform protein expression analysis in situations where they have very limited material (where they may never have been able to run a western before), patient material, mouse organs and cell lines. Common scenarios include looking to define a time point or a concentration dose for more focused studies, or studying a longer time course or dose range than you would normally have the capacity to perform westerns. We welcome generating focused collections for specific case studies.
More broad applications include:
- Predictive pharmaco-dynamics (PD) - Monitoring organ specific pathway effects of a compound in vivo
- Biomarker discovery/Translational sciences - Detection of disease relevant protein markers in human biopsy material, body fluids
- Drug combinations - Identify pathways of drug resistance and relapse (preclinical or clinical samples)
- Compound screening in cellular systems - EC50 value determinations to define on- and off-target effects to prioritize compounds
- Drug candidate profiling in vitro - pathway activity profiles in cell lines including patient-derived primary cells
- Infectious disease mechanism investigation - Pathway pertubation in host cells upon pathogen infection
- Genome-wide screens - RNAi or CRISPR/Cas to measure gene knock-down effects on activity of selected pathway nodes, couple with phenotypic profiling using high content imaging
Accessing the platform
The RPPA platform is open to researchers Australia-wide and can be accessed by connecting with A/Prof Kaylene Simpson as a first communication (see contact details below). The platform is extremely easy to use from a researcher perspective, you obtain lysis buffer from the RPPA team, create your lysates and send everything to the lab for running and analysis. We can provide a first pass analysis that documents QC metrics and limited statistical analysis such as fold change (depending on experimental design). You must lyse your cells in the lysis buffer designed for the platform, and we can exchange proteins to elute in this buffer if needed. The platform is highly sensitive so we recommend you perform 3 biological replicates for each sample to be analysed. If that is not feasible due to limited sample material we will discuss options with you. The platform has a detailed companion user guide and submission documents that must be filled out when submitting your samples.
Antibodies and services
We have curated over 200 antibodies (native and phospho isoforms) in at least 3 cell lines and to be included in the platform, antibodies must be the correct size and have limited additional bands. We can work with BYO antibodies and we offer an antibody curation service via western blotting. The platform is extremely flexible, you can choose any combination of targets to screen, or you can select some of our standardised library options that allow you to screen specific pathways and associated proteins.
How much protein do you need? Ideally we have 30ul of 2mg/ml protein concentration. Anything less than that means we are very creative in how we spot the samples, but we are happy to discuss if it is your only option.
Our antibody list is constantly evolving, if you don’t find what you are looking for, please don’t discount the platform, we have capacity to buy specific reagents so just ask!
The antibody list can be found here.
Please download the current pricing document.
- RPPA Team
A/Prof Kaylene Simpson – guidance on all aspects of assay development and interpretation
Arthi Macpherson – day-to-day RPPA operations: provision of buffer, sample management, instrument operation, analysis
Dr Twishi Gulati – assay development, RPPA operations, analysis reporting