Reverse Phase Protein Array (RPPA) Platform

The Reverse Phase Protein Array (RPPA) platform has been created in the laboratory of Cell Signaling, headed by Dr. Thierry Dubois, for a collaborative project with the pharmaceutical company Servier. The platform is open for collaborations since October 2009 thanks to funding from Cancéropôle Ile-de-France.

• Technique

RPPA (Reverse Phase Protein Array) is an innovative high-troughput proteomics technology that requires only very small amounts of biological material. This miniaturized dot-blot technology consists of depositing in an entirely automated manner cell or tissue lysates onto slides covered with nitrocellulose. Proteins of interest are subsequently detected using specific antibodies. Only about 1 ng of material is deposited per spot, which represents 10 000 times less than for Western Blotting, and up to a thousand samples can be analyzed simultaneously on the same slide. Using antibodies directed against modified proteins, such as phosphorylated proteins, RPPA can be used to analyze the activation status of specific cell signaling pathways.

Today, about 200 antibodies have been validated for their use on our platform. This panel of antibodies permits the study of all major signaling pathways. New antibodies are continuously being validated, according to ongoing scientific projects.

RPPA analysis requires only 25 µl at 1 mg/ml of total protein concentration per sample and is therefore particularly suitable for the analysis of large panels of precious samples, such as treatment kinetics, human biopsies, xenografts, flow cytometry-sorted cells, etc.

• Equipment

Since October 2009, we are equipped with an entirely automized spotting robot (Aushon Biosystems). This robot contains 16 tips of 185 µm in diameter, which transfer a volume of about 1.6 nl per sample, from 384 well sample plates to nitrocellulose-covered microscope slides. Used slides are Whatman FastTM or Schott Nexterion NC-W slides.

Antibody revelation on the slides is performed in an automated fashion using an Autostainer 48 LINK (DAKO). Fluorescence is detected using a scanner (GenePix 4000B) and signals are quantified with MicroVigene software (VigeneTech).

For data analysis, we collaborate with the bioinformatics group headed by E. Barillot (U900).

• Contact

For any further information, please contact us:
Leanne DE KONING, PhD
Institut Curie Département de Transfert
Plateforme RPPA
Hôpital Saint-Louis
Quadrilatère historique, Porte 13
1, rue Claude Vellefaux
75010 Paris
Tel +33 (0)1 53 19 41 34
Leanne.De-Koning@curie.fr