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Reverse Phase Array

Reproducibility of ERK detection using infrared dyes

Figure 1. 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).

Multiplexed detection of phosphorylated and total ERK protein levels

Figure 2. 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

Lysate (reverse phase) arrays contain complex samples, such as cell or tissue lysates, that are printed on an array surface and interrogated with antibodies4.

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

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