Protein Array

Application Overview

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.

^1.Sheehan, KM et al. Mol Cell Proteomics.4(4): 346-55 (2005)

^2.Loebke, C et al. Proteomics. 7(4):558-64(2007)

^3.Ambroz, K. et al. Poster presentation, Chips to Hit Annual Meeting (2005)

^4.Hall, DA et al. Mech Ageing Dev. 128(1):161-7 (2007)

[LEFT & BELOW] 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 IRDye800CW-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).

Multiplexed detection fo phosphorylated and total ERK protein levels

[LEFT] Figure 3. 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).

Workflow

[LEFT ] Figure 4. Schematic protocol for near-infrared lysate (reverse phase) arrays. Adapted from Calvert, VS et al. Clin Prot J. 1(1):81-89 (2004).

[ABOVE] Figure 5. Schematic of lysate and analytical protein array concepts. Adapted from Sheehan, KM et al. Mol Cell Proteomics.4(4): 346-55 (2005)