As in standard western blotting, proteins are size separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a blotting membrane ( Fig. The DigiWest approach combines standardized protein separation and western blotting with a multiplexed, bead-based immunoassay platform, such as the Luminex FlexMAP 3D system. An analysis of the expression of almost 200 proteins in tumour cells collected by laser capture microdissection from primary human mammary carcinoma demonstrates the capabilities of this approach for characterizing limited sample material.ĭescription of the DigiWest bead-based western blot We show that the sensitivity and reproducibility of this approach are as good as high-end western blotting systems and that the method is capable of providing high-resolution data on protein phosphorylation and expression. The digital nature of the resulting data prompted us to name this approach ‘DigiWest’. Accurate size information is obtained using western blot, and the advantages of a bead-based microarray platform-high throughput and low material consumption-are utilized to increase the output of this classical method. Here, we describe an approach that enables a highly parallel analysis of protein expression and modification status by adapting the classical western blot 13 to a bead-based microarray platform. High-throughput technologies often come with compromises in data quality, and highly reliable approaches such as western blotting are often required to subsequently confirm the results. This type of information helps provide a better understanding of cellular processes. Information on the presence of a wide variety of proteins can be obtained and, by detecting post-translational modifications, the activation states of regulatory cascades can be interrogated. Lower-throughput immunohistochemistry 10, high-content screening technologies 11 and reverse-phase protein microarrays (RPPAs) 12 allow for ‘systems biology approaches’ directly at the protein level. The proteome-wide generation of antibodies, driven by the Human Protein Atlas project 8 and other groups 9, opens up new possibilities and calls for immunoassay systems that are capable of performing multiple assays in parallel. By allowing the unbiased analysis of samples, mass spectrometry has had a massive impact on signalling research 6, as have immunoassay-based approaches 7. Although these breakthrough observations usually come from hypothesis-driven approaches, the development of new technologies 4 and advances in automation now result in new possibilities for understanding cellular signalling 5. Pathway dysregulation and aberrant cellular signalling have been linked to diseases such as cancer 2 and inflammatory disorders 3. Significant progress has been made in understanding cellular activity through the molecular analysis of signalling pathways 1. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. Dissecting cellular signalling requires the analysis of large number of proteins.
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