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PURETIME fluorescence polarization

PURETIME dyes have characteristically long fluorescence lifetimes. This makes them interesting as fluorescence polarization probes. Short lifetime fluors such as fluorescein, Rhodamine, Alexa, BODIPY or cyanine dyes produce highly polarised emission when attached to even modest sized biomolecules. PURETIME dyes do not emit highly polarised fluorescence until they are either in highly viscose environments or attached to proteins that rotate slowly in solution.

  

Applications

bulletMeasure changes in molecular weight at more than 40,000 Da
bulletMeasure changes in viscosity at more than 400 mPa.s
bulletDiscriminate against elastic scatter and reflection in challenging assay situations

 

Polarization characteristics

As an example, the figures below compare the fluorescence polarization of PURETIME 22 with fluorescein under similar conditions. All polarization measurements were performed with a BMG Fluostar platereader equipped with a polarising optical fibre head.

 

Perrin equation plot showing intercept at Pzero=400mP

 

Perrin equation plot showing intercept at Pzero=450mP

 

Although the two dyes have similar Pzero values,  PURETIME 22 doesn't plateau until attached to much higher molecular weight molecules as the Perrin equation plot shows below:

 

 

Perrin equation plot showing increased measurement window for PURETIME22

 

 

 

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Last modified: May 25, 2011