Antibody arrays for determination of relative protein abundances.

As a large number of genome-sequencing projects reached completion, the attention of the scientific community is turning toward understanding the structure-functions of gene translation products-the proteins as well as the complete complement of proteins-the proteome. One goal of proteomics is to correlate changes in protein abundance with biological processes and disease states. To help accelerate this avenue of proteomics, a significant effort has been devoted to the development of multiplexed methods for protein analyses. We have developed an Antibody Microarray, a chip-based technology for multiparallel determination of relative abundance of hundreds of proteins. The Antibody Microarray is composed of hundreds of distinct monoclonal antibodies printed at high density on a glass slide. It utilizes a novel experimental setup and data analysis algorithm, which enables scientists to assay hundreds of cytosolic, nuclear, and membrane-bound proteins with a single experiment. Examples of biological samples that are analyzed on the Antibody Microarray include tissue samples, cell cultures, and body fluids.

Multiplexing bioluminescent and fluorescent reporters to monitor live cells.

Reporter proteins are valuable tools to monitor promoter activities and characterize signal transduction pathways. Many of the currently available promoter reporters have drawbacks that compromise their performance. Enzyme-based reporter systems using cytosolic luciferases are highly sensitive, but require a cell lysis step that prevents their use in long-term monitoring. By contrast, secreted bioluminescent reporters like Metridia luciferase and Secreted Alkaline Phosphatase can be assayed repeatedly, using supernatant from the same live cell population to produce many sets of data over time. This is crucial for studies with limited amounts of cells, as in the case of stem cells. The use of secreted bioluminescent reporters also enables broader applications to provide more detailed information using live cells; for example, multiplexing with fluorescent proteins. Here, data is presented describing the characteristics of secreted Metridia luciferase and its use in multiplexing applications with either Secreted Alkaline Phosphatase or a fluorescent protein.

Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays.

In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems.