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Choosing & Preparing Biological Probes for FCS Assays

High quality fluorescently labeled probes are required for successful fluroescence correlation spectroscopy measurements. The following lists describe key probe properties to consider as you are designing FCS experiments.

Probe-Target Considerations
  • Affinity of Probe for Target: It is critical to understand the affinity of probe-target interactions in any FCS assay being conducted (See Biological Interactions that FCS Can Detect). Low affinity probes require high concentrations, while high affinity probes allow you to use low concentrations.
  • Selectivity of Binding: High sensitivity measurements require highly selective probes that only bind to the intended target. If there is non-specific binding, or even specific binding to small quantities of intefering substances(e.g. buffer components, carrier proteins such as BSA, sample chamber) in the sample, FCS results may be ambiguous.
Probe-Dye Considerations
  • Purity of the Probe:Probe purity will have a large impact on the quality of your FCS measurements. Probe samples should contain as little free dye as possible (See Choosing Fluorescent Dyes for FCS Assays). HPLC purification and a variety of spin purification schemes exist to ensure > 95% purity. Correlation analysis will easily detect free dye in excess of 5-10% of probe fluorescence. Although it is still possible to quantify FCS measurements made under these conditions, it does reduce the useful range over which assays can be conducted and may require more complex data analysis.
  • Specific Activity (dyes per molecule) of the Probe: The total number of dye molecules per labeled probe is the Specific Activity or Stoichiometry of labeled probes. This specific activity can be modulated to extend the useful range over which you can conduct FCS measurements using the QuantumXpert. In addition, knowing the specific activity enables you to quantify binding stoichiometry.
  • Affect of Dye Labeling on Probe Affinity: It is important to understand how labeling a probe affects binding affinity, especially when using it is used in competition binding experiments. If the biological activity of the probe is significantly altered by conjugation to a fluorescent dye (e.g. reduced affinity), it may not be ideal for use in highly sensitive FCS measurements.
  • Affect of Probe-Target Binding on Dye Quantum Yield: If quantum yield is altered when a labeled probe binds to its target, or when large complexes form, this must be accounted for when analyzing FCS data.