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Protein Array

Multiplexed detection fo phosphorylated and total ERK protein levels

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.
Reproducibility of ERK detection using infrared dyes

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).



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