Reverse Phase Proteomics Array

Description of RPPA

undefinedReverse-phase protein array (RPPA) is a fantastic high throughput counterpart to the mass spectrometry-based proteomics approach. Based on a dot blot approach, picolitres of a sample of interest are spotted on a nitrocellulose membrane. With 10,000 simultaneous assessments possible on a single microscope slide, the throughput of RPPA is impressive. More typical runs involving 60 or a few hundred samples allowing scope for technical replicates and dilution curves to be printed on the same array, all of which can then be incubated with primary and secondary antibodies simultaneously, allowing for superior and statistically validatable quantification.

Our Platform Pipeline

Consultation

Initial interest in the utilisation of the RPPA platform can be expressed by email at RPPA@lih.lu or by a phone call to Joseph Longworth, the platform head. During the consultation, we can provide feedback on the optimal use of the platform and design of the project. We can also discuss which of our antibody targets are of interest to your project, as well as the potential for inclusion of new targets into the platform. We will also review with the user the sample preparation procedures within their project and the timelines expected for sample provision and processing. Once finalised, more accurate pricing can then be provided. 

Sample preparation

Once the project is finalised and the samples provided, samples will be lysed and quantified before diluting appropriately ready for printing. Printing is performed with our GeSIM Nano-Plotter 2.1 onto nitrocellulose slides. Gaskets facilitating up to 16 antibodies per slide, depending on the number of samples, will then be attached and an antibody-based quantification performed using our InnoScan 710 Microarray scanner.

Bioinformatics

Your samples will then be processed through our bioinformatic pipeline and the raw data along with pathway and ontology linked analysis will be fed back to the user. Additionally, we have excellent links with the bioinformatics platform who can be contacted for deeper project-specific analysis of the data (quoted for by the bioinformatics platform).

Validation

As with any antibody-based assay including  ELISA and immunohistochemistry, RPPA is sensitive to the specificity of the antibody to the target of interest. We endeavour to utilise only the highest quality of antibody, most of which have previously been validated either in-house or in other RPPA facilities. We can support you in any further validation required for the antibodies of interest within your sample type or with our close ties to the proteomics platform co-located with the RPPA facility.