Hi folks,
I am planning to synthesize a set of FRET substrates and figured that I have good FRET pairs in stock as NHS esters. I am aware several protocols are available for the purification of labeled proteins (gel-filtration, dialysis, etc) but weight cut-off really don't help that much with short peptides (MW being around 1500 Da).
I thought it would be wise to synthesize the peptide substrate by adding specific lysines already conjugated with the fluorophore and quencher...but have no idea how I would purify conjugated Lys from free dyes either.
Any thoughts?
Really appreciate your help!
Thanks!
Use HPLC if the dye is very water soluble(for example, dye with SO3H), use silica gel chromatgraphy if the dye is not water soluble (no SO3H group).
I have used chromatography on Sephadex LH-20 in water to separate labeled peptides from free dye and unlabeled peptide. This obviously requires that the product be water-soluble, but the same method can be used with some organic solvents or organic/aqueous mixtures if necessary. It's basically an isocratic reverse-phase separation, but doesn't require any equipment other than a glass column and can easily be scaled up. HPLC will of course give a much higher separation efficiency, if the equipment is available.
For water soluble dye conjugate, a reverse phase C18 column may do the separation as well for mixture with decent separation! Use a gradient of water/acetonitrile (or methanol), start from proper low CH3CN% (or 0%) to higher , depends on the hydrophilicity of your compounds.
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