Protocol

Protein Arrays: Sandwich Approach for Analyzing Complex Solutions

This protocol was adapted from "Characterization of Protein Complexes," Chapter 10, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Protein arrays can be used to quantitate the abundance and modification states of proteins in complex mixtures. For this application, it is necessary to array-capture reagents, such as antibodies, on the slides and then use these molecules to specifically capture their cognate antigens from solution. The investigator has a choice in either labeling the proteins that are under study or detecting the captured proteins by an indirect labeling method. In the latter case, which is detailed in this protocol, each protein is captured by one antibody (or other capture reagent) and then detected in a second step with a second reagent. The second reagent, which in this case is also an antibody, recognizes the protein at a site that does not overlap with the recognition site of the capture reagent. In this sandwich approach, the second reagent is labeled and so provides the signal. The advantage of this approach is that it does not require labeling the proteins themselves. Moreover, additional specificity is gained by using two different reagents for each protein. The disadvantage, however, is that it is more difficult to assemble matched pairs of antibodies for each protein of interest.