New 3D Black Substrate for Protein Microarrays with Improved Dynamic Range
Poster Jan 03, 2006
Markus Hollas, Eric Jallerat, Karl Pflanz, Inka Praulich, Johanna Walter
IntroductionSurge in proteomics research has accelerated the development of new protein arrays as an indispensable tool to study high density protein interaction. The most important factor that determines the performance of protein microarrays is the substrate on which the proteins are immobilised. The microarray substrate, used either in an interaction or capture mode has to provide several key functions. High binding capacity for any proteins without changing their biologically active conformation as well as a low auto fluorescence in order to generate a high signal to noise ratio are essential. This enables the detection of the interaction or capture event within a widedynamic range.
A Microarray slide combining the benefits of increased surface area of 3D cellulose nitrate with the excellent low auto fluorescence of a black substrate has been developed and evaluated. The 3D structure was designed to offer a high binding affinity for proteins and to better stabilize their active conformation even in presence of denaturing temperature conditions. A competitive study analysing the background fluorescence and the detectable concentration range of a chosen antigen is presented. An Affymetrix Spotting roboter 417 arrayer was used for protein dotting. The analysis is based on fluorescence scanning experiments with an Affymetrix 428 array scanner at a gain of 60. Data analysis was made with ImaGene Premium 5.5.3 by Biodiscovery Inc.
When there is a need to quickly analyze samples using a number of different PCR assays, it is likely that optimal conditions for each assay will not be the same. First, different assays often will require different annealing temperatures for their primers. In addition, amplicons may be designed to be of different lengths and therefore require varying durations of the extension step.READ MORE