What factors should be considered when selecting dyes?
Dyes can be purchased from Molecular Probes, Inc., (Alexa), Atto Tec, Siegen, Germany (ATTO 550 and ATTO 647) or Amersham (Cy 3 and Cy 5). Dyes of course differentiate in their quantum efficiency, stability against oxidation and bleaching, reactivity and quality. Ideally one will use dyes that have a comparable signal yield for two colour experiments. We personally have good experiences with ATTO dyes in this regard. Crucial for the quality in labeling experiments is the purity and the activation degree (NHS-ester) of the dyes. Both factors influence the background and signal intensity. Important is to store the dyes in water free solvents. Amino groups are far more reactive with the NHS ester than water (more than 1000fold). Thats why the labeling can be done in buffered solutions.
Dyes can be purchased from Molecular Probes, Inc., (Alexa), Atto Tec, Siegen, Germany (ATTO 550 and ATTO 647) or Amersham (Cy 3 and Cy 5). Dyes of course differentiate in their quantum efficiency, stability against oxidation and bleaching, reactivity and quality. Ideally one will use dyes that have a comparable signal yield for two colour experiments. We personally have good experiences with ATTO dyes in this regard. Crucial for the quality in labeling experiments is the purity and the activation degree (NHS-ester) of the dyes. Both factors influence the background and signal intensity. Important is to store the dyes in water free solvents. Amino groups are far more reactive with the NHS ester than water (more than 1000fold). That’s why the labeling can be done in buffered solutions.
