Dendra activation

[David Stanek-3]

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We use the photoconvertible Dendra protein to tag different proteins,  
photoconvert it from green to red using 405nm laser (we never managed  
to convert it using 488nm) and trace movements of the tagged  
proteins. However, sometimes in addition to green-red  
photoconversion  we also observe increased fluorescence of the  
original green variant. Does anyone have a similar experience and  
solved that problem?
Many thanks
David

[John Runions]

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Hi David,

Can it be that the emission spectrum of the converted form is broad and
cross-talking at the lower end into your green detection region?

John.


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We use the photoconvertible Dendra protein to tag different proteins,
photoconvert it from green to red using 405nm laser (we never managed
to convert it using 488nm) and trace movements of the tagged
proteins. However, sometimes in addition to green-red
photoconversion  we also observe increased fluorescence of the
original green variant. Does anyone have a similar experience and
solved that problem?
Many thanks
David



*********************************
C. John Runions, Ph.D.
School of Life Sciences
Oxford Brookes University
Oxford, UK
OX3 0BP

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[Ian Dobbie-2]

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John Runions <[hidden email]> writes:

>> We use the photoconvertible Dendra protein to tag different proteins,
>> photoconvert it from green to red using 405nm laser (we never managed
>> to convert it using 488nm) and trace movements of the tagged
>> proteins. However, sometimes in addition to green-red
>> photoconversion  we also observe increased fluorescence of the
>> original green variant. Does anyone have a similar experience and
>> solved that problem?
>> Many thanks
>> David
>
> Can it be that the emission spectrum of the converted form is broad and
> cross-talking at the lower end into your green detection region?
>
> John.

Alternatively if you have very high concentrations of unconverted
fluorophore it might be self-quenching. When a fraction is converted
to the red form the concentration of green fluorophore decreases and
produces less self quenching, hence (unintuitive) increasing both the
green and the red signal.

Ian