Long-Lifetime FP Labels:

For a demonstration of the behavior of a short lifetime probe vs. a longer lifetime probe in a Fluorescence Polarization Immunoassay (FPIA) we use the example of an FPIA of HSA (MW ~ 60 KDa):

On one side HSA (the antigen) is labeled with SeTau 425 a long-lifetime label with a lifetime of 26 ns lifetime (left) and on the other side (right) fluorescein, a commonly used 4ns lifetime label. In the case of SeTau 425 the labeled HSA has still a relatively low starting polarization of 165 mP and only upon addition of the specific antibody the rotational mobility changes and as a result the polarization increases. A different behavior is shown with fluorescein-labeled antigen (right): The labeled antigen has a polarization of 130 mP. Typically one would expect a much higher value but the lower polarization is due to the fact that several fluorescein molecules are labeled on one HSA molecule and undergo homo-ET. Due to homo-FRET the polarization value for the conjugate decreases. Nevertheless, the important observation is, that there is neither a polarization increase observed upon titration with the specific (red) nor non-specific (blue) antibody due to the fact that the lifetime of fluorescein (τ = 4ns) is much shorter than the rotational correlation time (Θ = 40 ns) of the antigen.

Applications

Measurement of high molecular-weight analytes (20 – 80 kDa) in a fluorescence polarization assay (FPIA). For more specific data we refer you to the specs sheets and the table below.

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Red Excitable FP-labels:

SETA BioMedicals also offers several red excitable dyes that have adequately long lifetime to be used in FP assays for low molecular weight analytes. These labels have unmatched sensitivity and photostability. An additional advantage is the large Stokes’ shift in the order of 45 – 50. For more specific data we refer you to the specs sheets and the table below.

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DNA hybridization reactions can be easily monitored by means of fluorescence polarization using the newly developed fluorescence labels from SETA BioMedicals. Square 660 NHS is a fluorescent label that is easily coupled to amino-modified oligonucleotides. It is a very sensitive fluorescent polarization label with absorption and emission maxima and the NIR range. Covalent attachment to an oligonucleotide results in an about 10-fold increase of its fluorescence lifetime (lifetime for labeled oligos are in the order of 3 ns) and thus lower initial polarization of the labeled oligonucleotide. Interaction of a dye-labeled oligonucleotide (see below) with the complementary oligonucleotide or DNA leads to a significant change in fluorescence polarization due to the increase in mass. Importantly in case of Square 660 binding of the complementary strand or DNA is accompanied also by a reduction in lifetime (from 3 ns to 1.8 ns) which further should help to increase the dynamic range of the assay. Based on the increased lifetimes of Sq 660 (K8-1352) in presence of oligonucleotides this dye is perfectly suited as a label for in-situ hybridization assays using fluorescence polarization as the read out parameter.

Please contact us for more information.

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