Abstract
Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. 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 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. 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.
Highlights
Dissecting cellular signalling requires the analysis of large number of proteins
These breakthrough observations usually come from hypothesis-driven approaches, the development of new technologies4 and advances in automation result in new possibilities for understanding cellular signalling5
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
Summary
Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. 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. 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. We describe an approach that enables a highly parallel analysis of protein expression and modification status by adapting the classical western blot to a bead-based microarray platform. 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
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