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Fluorescence Polarization

Fluorescence polarization (FP) measurements are based on the assessment of the rotational motions of intrinsically or extrinsically fluorescent molecules in solution. FP is the result of the competition between the molecular motion and  fluorescence lifetime of an ensemble of fluorescent or fluorescently labelled molecules in solution. It is a versatile solution-based technique that has been widely utilized to study molecular interactions, enzymatic activity, and nucleic acid hybridization. Several reporter properties such as fluorescence lifetime, linker length, hydrophobicity/hydrophilicity, quantum yield, and extinction coefficient need to be considered when developing FP assays. Fluorophore lifetime, antigen size and molecular weight changes upon binding determine the dynamic range of FP assays.

For a general overview on FP we refer you to the following technical note (FP).

Excitation Light Sources Characteristics
Product Number
(Specs Sheet)
Product Name
(Product Info)
Target Group 380 405 436 488 532 594 635 650 680 700 750 780 Medium λ abs
[nm]
ε
[M –1
cm–1]
λ em
[nm]
QY
[%]
FLT
[ns]
Buy
                   
K1-204
K1-204
SeTau-380-NHS
SeTau-380-NHS
NH2 Water 270 23800 480 56 32.5
K5-3222
K5-3222
Seta-580-NHS
Seta-580-NHS
NH2 PB 7.4 578 90000 602 55
K7-547
K7-547
SeTau-405-NHS
SeTau-405-NHS
NH2 PB 7.4 405 13800 518 80 9.3
K7-567
K7-567
SeTau-405-Azide
SeTau-405-Azide
triple-CC PB 7.4 405 13800 518 80 9.3
K7-545
K7-545
SeTau-425-NHS
SeTau-425-NHS
NH2 PB 7.4 425 4200 545 39 26.2
K8-1352
K8-1352
Square-660-NHS
Square-660-NHS
NH2 PB 7.4 657 182000 676 3 0.27
K9-4119
K9-4119 new
SeTau-665-NHS
SeTau-665-NHS
NH2 PB 7.4 664 160000 712 53 3.1
K9-4148
K9-4148
SeTau-647-Maleimide
SeTau-647-Maleimide
SH PB 7.4 648 200000 692 45 3.2
K9-4149
K9-4149
SeTau-647-NHS
SeTau-647-NHS
NH2 PB 7.4 649 200000 695 61 3.2
K9-4169
K9-4169 new
SeTau-670-NHS
SeTau-670-NHS
NH2 PB 7.4 673 275000 694 36 1.6
K8-1671
K8-1671
Seta-646-Maleimide
Seta-646-Maleimide
SH PB 7.4 647 210000 657 8
K7-548
K7-548
SeTau-405-Maleimide
SeTau-405-Maleimide
SH PB 7.4 405 13800 518 51 9.1
KS-550
KS-550
510–560 nm FP Standard
510–560 nm FP Standard
Water 549 567
K8-3003
K8-3003 new
Seta-470-Maleimide
Seta-470-Maleimide
SH Water 469 50000 521 1.9
K7-544
K7-544
SeTau-425-Maleimide
SeTau-425-Maleimide
SH PB 7.4 425 4200 545 39 26.2
K8-1389
K8-1389 new
Seta-700-NHS
Seta-700-NHS
NH2 PB 7.4 688 162000 703 14

Several different types of fluorescence polarization labels are available for homogeneous FP assays.

SeTau FP labels exhibit lifetimes from 9 to 32 ns and are suitable for measurement of antigens with molecular weights in the range between 10 and 80 kDa. This MW-range has not been addressed with common polarization labels or metal-ligand complexes (MLCs) based on Ru, Os or Re.

The superior red-excitable FP labels (e.g. SeTau-647 or Square-660) have lifetimes in the order of 3 ns and are very photostable.

 

Fluorescence polarization labels: fluorescence lifetime (FLT) versus molecular weight of analyte

 

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 ~ 66.5 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) with fluorescein, a commonly used 4 ns 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 seen with the 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-FRET. Due to homo-FRET the polarization value for the conjugate decreases. Nevertheless, the important observation here 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 = 4 ns) is much shorter than the rotational correlation time (Θ = 40 ns) of the antigen.

Fluorescence polarization of Seta-425 bound to anti-HSA and IgG

SeTau-425

τ = 26 ns

Fluorescence polarization assay with long-lifetime dye SeTau-425
Fluorescence polarization of fluorescein free bound to anti-HSA or IgG

Fluorescein

τ = 4 ns

 

Applications

Measurement of high molecular-weight analytes (10 – 80 kDa) in a fluorescence polarization assay (FPIA). For more specific data we refer you to the specs sheets and the table below.

 

Product Number
(Specs Sheet)
Product Name
(Product Info)
Lifetime at 25 oC
[ns]
K1-204 SeTau-380-NHS 32.5
K7-547 SeTau-405-NHS 9.3
K7-545 SeTau-425-NHS 26.2

 

Red Excitable FP-labels:

Red excitable dyes with longer lifetimes between 1.6 - 3 ns and larger Stokes' shifts in the order of 21 - 50 nm for use as labels in FP assays are shown in the table below. These labels have unmatched sensitivity (extinction coefficients of  up to 275,000 M-1.cm-1 and Q.Y.s up to 60% in aqueous solutions) including very high photostability. SeTau-647 and SeTau-670 are more sensitive labels for polarization measurements compared to Alexa 647, Alexa 660 or Cy5!

For more specific data we refer you to the specs sheets in the product table below.

 

Product Number
(Specs Sheet)
Product Name
(Product Info)
Lifetime at 25 oC
[ns]
K9-4148 SeTau-647-Maleimide 3.2
K9-4149 SeTau-647-NHS 3.2
K9-4169  SeTau-670-NHS 1.6
 

 

Measurement of DNA-oligonucleotide binding:

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 a low initial polarization of the labeled oligonucleotide. Interaction of the 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 and reduction in lifetime of Square 660 (from 3 ns to 1.8 ns) upon binding of the complementary strand or DNA, which further helps to increase the dynamic range of the assay. Based on the increased lifetimes of Square-660 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.

Fluorescence polarization assay (FPA) of Square-660-oligo demonstrating polarization increases upon binding to the complementary oligonucleotide or DNA

Fluorescence polarization assay (FPA) of Square-660-oligo
demonstrating polarization increases upon binding to the
complementary oligonucleotide or DNA

 

 

Product Number
(Specs Sheet)
Product Name
(Product Info)
Lifetime at 25 oC
[ns]
K8-1352 Square-660-NHS 3.0

 

 

 

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