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April 15, 2011 (Vol. 31, No. 8)

Combining Labeled and Label-Free Tools

Advantages Include a More Complete Picture of Intricacies of Cellular Systems

  • Maximizing Useful Data

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    Figure 2. Cell cytotoxicity using label-free technology before and after receptor activation

    Figure 2 demonstrates the noninvasive nature of label-free technology. The PerkinElmer ATPlite® cytotoxicity assay results, demonstrated that the EnSpire label-free platform had no effect on cell viability. ATPlite was run on endogenously expressed receptors in cells that had undergone a 60-minute label-free scan, both pre- and post-receptor activation, and also run on identical cells that did not undergo label-free analysis. There was no measurable cytotoxicity relative to the positive control showing that cells can be re-used after label-free analysis.

    This means that one batch of cells can be used to run two assays. As production of cells is both a laborious and costly step in the research workflow, running label-free assays combined with, for example, luminescence or fluorescence assays, using the exact same batch of cells is a real benefit.

  • SRA Analysis

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    Surface plasmon resonance (SPR) is a label-free technology that has proven powerful in characterizing protein:ligand interactions. Interacting species are immobilized onto a surface and as interaction partners bind to immobilized molecules, refractive index at the surface alters in proportion to the change in mass concentration. Changes in SPR signal over time are then presented graphically.

    The results can be used to display the formation and dissociation of complexes over the entire course of an interaction, with the kinetics (association (ka) and dissociation (kd) rates) shown by the shape of the reaction curve. The affinity (binding strength KD) of the interaction is also an available output of this technique.

    Optical label-free generates comparable results to SPR for binding strength. This offers an exciting opportunity to use the two techniques in a complementary way. Our experiments have shown how molecular interactions between carbonic anhydrase and a number of low molecular weight molecules were reliably detected with picometer shifts down to around 10 pm.

    Furthermore when KD values were calculated and compared to values determined by a market-leading SPR system, the values were in broad agreement across the molecular weight range as shown in the Table.

    When nonplate based SPR becomes a throughput bottleneck, the EnSpire with Epic label-free technology can help as it’s a lower cost plate-based system. By using the Epic technology to screen based on binding strength, (KD), the compounds that demonstrate binding can progress into SPR for determination of ka and kd rates. This lower sample number becomes manageable for the SPR system. The two systems are complementary.

    In summary, combining optical label-free technology with classical labeled methods gives a more complete picture of cellular systems, while combining different types of optical label-free technology such as Epic technology and SPR together can bring throughput and workflow benefits to biochemical protein:ligand binding, thus helping to drive research forward.

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