I want to use fluorescent anisotropy(polarization) to detect protein-DNA binding. The signal of tryptophan is different with or without DNA binding. I know that PTI QuantaMaster spectrometer can do this, but it is unavailable for me. Can I use some other equipment?
ps.
As far as I know, by using fluorescence anisotropy, I need to use an excitation wavelength of 295 nm to selectively excite tryptophans and emission detection at 344 nm.
You could try to use Circular Dichroism choosing a wavelenth were there is only the contribution of one of the pateners of the binding. You can do the spectra of the protein and the DNA separately and find the best lamda to follow the binding process
i had once used Perkin Elmer LS 55 Fluorescence Spectrometer for studying DNA-small molecule, and later prot-prot interaction. http://www.perkinelmer.com/IN/Catalog/Product/ID/L2250107
You could try to use Circular Dichroism choosing a wavelenth were there is only the contribution of one of the pateners of the binding. You can do the spectra of the protein and the DNA separately and find the best lamda to follow the binding process
Thank you sabyasachi and concetta. It is a pity that Perkin Elmer LS 55 Fluorescence Spectrometer is also not available for me.
As for the CD method, can it fit the kd of the protein-DNA complex? How is the accuracy? Attached papers are appreciated.
I send you a chapter of a book that can be useful for the binding analysis (see the paragraph 2.2.3)
For spectral analysis you can use any fluorescence spectro having polarizer and analyzer attachment. But i think CD analysis will be better for any kind of analysis regarding protein. If you wish to calculate binding energy you can do fluorescence polarization from there you can get a derived data of binding energy. But i will suggest you to go with ITC.
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