Testing and Estabilishing Optimal Protein Array Conditions to Be Applied towards the Darpp-32 Phosphoprotein Model System

Please Note: The research described below represents a small portion of an ongoing technical development project. Any and/or all of the methods used for this project may or may not be implemented in the final NIDA Neuroproteomics Center microarray protocols. ABSTRACT The ability to quantitate multiple proteins simultaneously has a wide array of applications including basic biological research, identification of therapeutic markers and targets, molecular classification and disease diagnosis. The challenge for microarray fabrication becomes one of choosing and optimizing conditions for printing and using the arrays that are robust enough to be useful for many diverse proteins. The major parameters that will be addressed include surface chemistry for printing; spotting method; protein concentration for printing; blocking and conjugation method; and signal generating molecule and labeling. The data obtained will be used to formulate standard operating protocols for the DARPP-32 array. INTRODUCTION Classical approaches to biological research have involved the study of one or a few genes at a time. Gene expression analyses at the mRNA level were completed using techniques such as Northern blotting, differential display, serial analysis of gene expression (SAGE), and dotblot analysis (van Hal et al, 2000). For the past decade, emphasis has been placed on generating DNA sequence information as a key to solve various biological problems and to gain a better understanding of the biological system.