Three principal MWA platforms have been developed to accommodate most currently envisioned study types:
A. 1- 6 samples, 96 well
B. 7-16 samples, 48 well
C. 18-180 samples, 4 wells
The exact number of samples and protein targets tested will depend on if and how the client elects to perform technical replicates and other experimental design considerations. Illustrations of the three main formats are below.
Small sample number & high number of protein targets
This approach is designed to examine cellular signal transduction networks from perturbation time courses (i.e. growth factor, cytokine, or hormone stimulation). 32-192 protein targets can be analyzed in up to six samples (e.g. time points, experimental treatment conditions, etc.) per blot depending on the number of desired sample replicates (see fig. 1 below for reference).
Figure 1 (left). A431 skin carcinoma cells were stimulated by EGF growth factor at six time points (0, 1, 5, 15, 30, and 60 minutes), lysed, and printed 96 times in an 8 X 12 micro-well format. Following blot transfer, 96 antibodies (red) and a loading control (? actin, green) were applied (modified from Ciaccio et al., Nature Methods, 2010).
Medium sample number & medium number of protein targets
Designed for studies of comparative analysis, this approach is suggested for hypothesis-driven experiments assessing two or more biological sample types (i.e. cell lines, tissues) and/or perturbation conditions. 16 – 96 protein targets can be analyzed from up to 16 samples per blot (fig. 2).
Figure 2. SkBr3 and T47D breast cancer cells were stimulated with EGF growth factor at six time points (0, 1, 5, 15, 30, and 60 minutes), lysed and printed side-by-side in technical triplicate. 16 antibodies were applied to the blot including phospho-MEK and Tubulin (above). 96-well gasket apparatus shown for reference (bottom).
High sample number & low to high number of protein targets
This method is intended for studies incorporating larger sample numbers (e.g. tissue/tumor micro-dissections, NCI60 cancer cell lines, antibody validation, etc.) with low to high numbers of protein targets. 4-8 proteins can be examined from 60-180 samples per blot. This approach is particularly attractive for studies from limited sample quantities as the MWA method requires only nanograms of cellular material (which is about two hundred fold less than a traditional western blot) per protein analyzed (fig. 3).
Figure 3 (above). Nine breast cancer cell lines subjected to control and growth factor conditions were printed and assayed via MWA in triplicate. Shown above is phospho-EphrinB1 (Y324/329).
MWA sample input versatility
In addition to a growing number of cell lines, the MWA approach/formats have also been successfully applied to tissue and whole organism target screens (fig. 4 below).
Figure 4 Left panel Infarcted mouse cardiac tissue assayed for tropomysoin expression. Middle panel Whole D. melanogaster varying in protein expression by developmental stage. Right panel Human breast tissues of diverse genetic profiles assayed for phospho-HER2 expression .
Microwestern Array Core Facility (MWAC)
The University of Chicago
Knapp Center for Biomedical Discovery
900 E. 57th St. Chicago, IL 60637