To amplify or not to amplify: A comparison between fluorescent detection methods for protein microarray applications

Protein microarrays are the template of choice for many diagnostic applications and proteomic analyses. Reverse Phase Protein Arrays (RPPA) enable the protein content analysis of hundreds of samples in a single assay. Furthermore, picoliter amounts of samples are most often sufficient to provide a significant signal for a given factor. These advantages make RPPA an attractive approach for biomarker evaluation in large patient cohorts. Despite similar functional features with DNA microarrays, a specific substrate composition is essential for protein-based microarrays. Indeed, cellulose nitrate membrane casted on a glass object is most often the substrate of choice for RPPA because of its high protein binding capacity. Since protein expression of a given factor can vary from femto- to micro-molar concentrations, detection sensitivity and dynamic range are two highly critical points to achieve reliable results. Even though signal amplification methods have been successfully used to improve the signal detection levels, it is often accompanied by a narrower dynamic range because of signal saturation by highly expressed proteins. In this work, we demonstrate the influence of the signal detection wavelength in combination with Innopsys dynamic range extension (XDR) detection method on both the sensitivity and dynamic range of the fluorescent signal measured with or without signal amplification on UniSart® Microarrays slides. Whereas the signal amplification reaction seems to be beneficial when measuring at 670nm, it can be circumvented to detect the same protein levels when measuring at 785 nm. Moreover, scanning in a XDR mode allows the use of signal amplification reagents to increase assay sensitivity while keeping 6log dynamic range at 785 nm. Taken together, the choice of fluorophore and scanning method can largely influence the quality and intensity of the measured signals.


Adriana Lagraulet1, Olga Böhm3, Eric Jallerat3, Susanne Drebing3, Marie-Laure Schneider2 and Denise van Rossum3

  • 1. Innopsys, Parc Activestre, Carbonne – France –;
  • 2. Innopsys Inc. 3440 S. Dearborn St, Ste#1035, Chicago IL 60616 – USA –
  • 3. Sartorius- Stedim-Biotech, August-Spindler-Str. 11, 37079 Gottingen, Germany; eric.jallerat@SARTORIUS.COM


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