Figure 1. Multiplexed detection of phosphorylated and total ERK protein levels. Tissue samples were obtained for three human breast cancer patients; lysates were prepared from these and from a Jurkat T cell control. The array was probed simultaneously with antibodies against phospho-ERK (mouse) and total ERK (rabbit), and detected with near-infrared labeled secondary antibodies (red, green). Overlaid image of total and phospho-ERK levels is shown at the top (red + green = yellow). Adapted from Calvert, VS et al. Clin Prot J 1(1):81-89 (2004).
The protein array is a higher-throughput way to generate information about protein abundance or modification state.
Using IR fluorescence to detect your protein arrays will give you:
- Dramatically reduced background autofluorescence of nitrocellulose-coated slides1
- Sensitivity in the femtogram range2
- Wide dynamic range
- Multiplexed detection to quantify and compare two targets3
- Simplified detection protocol3
Common types of protein arrays4 include:
- Lysate (reverse phase) arrays contain complex samples, such as cell or tissue lysates, that are printed on an array surface and interrogated with antibodies. Advantages include:
- More quantitative and reproducible than Westerns
(intra-chip variation ~0.1%2)
- Wide dynamic range
- Conserve precious samples
- Very sensitive — can detect femtogram or single-cell protein levels2
- Spot recombinant protein standards for absolute quantification2
- Run many replicates and dilutions easily
- Analytical arrays use affinity reagents such as antibodies or peptides to profile analytes in complex mixtures of proteins. These arrays can be spotted on a chip or slide, or spotted into microwell plates. Antibody capture arrays are the most common form.
- Example: Quansys ELISA arrays
- Functional protein arrays are spotted with many different purified proteins, and used to assay biochemical functions such as protein-protein, protein-DNA, protein-small molecule interactions and enzyme activity.
Figure 2. Reproducibility of ERK detection using infrared dyes. Lysate was prepared from a human breast cancer tissue sample. It was arrayed in triplicate in a series of two-fold dilutions, and stained with rabbit anti-ERK primary Ab and IRDye® 800CW dye-labeled goat anti-rabbit. Linearity and reproducibility of this data is shown in the graph. The average of three replicates is plotted; error bars are included but are very small and difficult to see. Adapted from Calvert, VS et al. Clin Prot J 1(1):81-89 (2004).