Covalent Labelling of Proteins and DNA

The factors required for a dye to act as a good covalent fluorescent label have been defined. Some of the chromophore dependent issues are discussed below. 

Brightness. Brighmess of a fluorophore is proportional to the product of the molar absorption coefficient at the excitation wavelength times its quantum yield. This is the theoretical value, but in practice it can be much reduced by fluorescent quenching on interaction with other labels on the protein or DNA surfaces. Sulfonic acid groups on the aromatic rings of cyanines reduce this interaction, giving very much improved protein fluorescence. 

Labelling Chemistry and Sequencing. The two main groups for labelling proteins are amine reactive and thiol reactive. For reaction with the amino groups in proteins the fluorophore is converted into the succinimidyl ester derivative by reaction with iV-hydroxysuccinimide, e.g. (3.74), and for reaction with thiols group this is achieved by making iodoacetamide or maleimide derivatives, e.g. (3.75). 

Photochemical Stability and the Wavelength of Excitation. Cyanines of chain length beyond Cy3 snffer from increasing photochemical instability. This can be a problem when they are nsed in conjunction with solid-state lasers operating around 630-650 nm. To overcome these problems workers at Boehringer Mannheim have developed the so-called pentacyclic fluorescent labels based on either the oxazine or rhodamine ring systems, e.g. Light Cycler Red 640 NHS (3.76).  

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