Pd 10 sephadex g 25 m

Atto655 labeled streptavidin was purchased from AttoTec GmbH (Siegen, Germany). Alexa 555 labeled SA was prepared in our own lab as follows: Streptavidin was labeled via N-hydroxysuccinimid (NHS)-ester conjugate Alexa555 (Lifetechnologies, Vienna, Austria) according to protocols provided by AttoTec GmbH (Siegen, Germany). For the labeling, 30 μl of a 1 mg∕ml streptavidin (dissolved in PBS buffer) stock solution were adjusted to a pH of 8.6 with a 0.2-M sodium bicarbonate buffer. A labeling ratio of 1:3 streptavidin to fluorophore has been adjusted. The reaction mixture was incubated for 1 h at room temperature. The labeling solution was purified via a PD-10 Sephadex® G-25 m (GE Healthcare, Pittsburgh) purification column in PBS buffer. To concentrate the labeled streptavidin, the solution was transferred to a cut off filter (3–10.000 MWCO (Molecular Weight Cut Off) PES) and centrifuged at 1200 rpm in a centrifuge (Thermo, Multifuge 1S-R, HERAEUS, Thermo Scientific, Vienna, Austria) until only 100–200 μl remained. The concentrated solution was split into aliquots and stored at −20°C.