Dear All,
I am planning to study Protein-DNA interaction in my research work. What I am planning to do is to carry out EMSA experiment but non radio labelled one! So, what comes in my mind is to tag either Cy3 or Cy5 onto the 5'end of Oligos.
Is there any one here who has done similar kind of work or had an experience in doing EMSA studies with similar kind of dye. This is the first time I am planning to do so please help me in deciding which one I should go for it. Cy3 or Cy5??
My colleague are suggesting Cy3 do well!
Looking forward for all of your valuable suggestion.
Thanks
John.
You should be able to use either one, but they have different excitation and emission wavelengths, so make sure your imaging equipment has the right filter set.
Adam is right. That will depend on your equipment.
Cy3 is excited by the green laser (530nm), which is not very common, although it can be excited by the blue laser (488nm) but with a weaker emission and you need a filter around 575nm.
Cy5 is excited by the red laser (640nm), which is much more common, but you'll need a filter around 660nm.
You should prefer the Cy5 of far red region to reduce the internal fluorescence.
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