Nap 5 sephadex column

The FO-SPR instrument and accompanying sensors were manufactured as described by Knez et al. [14 (link),21 (link)]. 5′ or 3′ thiol functionalized hybridization probes were first activated with dithiothreitol (DTT, 0.1 M in Phosphate Buffer (PB) 0.18 mM, pH 8.3). DTT was used to split thiol dimers which could prevent the surface functionalization process and it was removed from the solution by DNA purification with a NAP-5 Sephadex column (GE Healthcare, Oslo, Norway). Those 5′ or 3′ thiol functionalized hybridization probes were immobilized on the Au NPs or FO-SPR sensors by adding 1 µM of probes. An accelerated salt maturation protocol was used to maximize the DNA density on the Au NPs. [29 (link)] Au NPs were then washed three times in PB with 0.01% SDS and stored in the same buffer at 4 °C until further use. Both the FO-SPR sensors and Au NPs were backfilled to enable the PCR assays by incubating them in a 50 µM alkane thiol PEG (Polypure, Oslo, Norway) dissolved in pure ethanol for 2 h. Finally, both surfaces were washed three times with a 0.01% SDS PB buffer and stored afterwards at 4 °C in H₂O until further use.

The monoclonal mouse antibody TuBB-9 was produced by hybridoma cells, kindly provided by the Leibnitz Research Center Borstel, Germany. Hybridoma cells were cultivated in bioreactor cell culture flasks (INTEGRA Biosciences, Switzerland). TuBB-9 was purified from the culture supernatant with protein G columns. For labeling with FITC (fluorescein 5(6)-isothiocyanate, Sigma, USA), TuBB-9 (1 mg/ml in sodium carbonate buffer at pH 9.3) was mixed with FITC (2.57 mM in DMSO) in a molar ratio of 20:1. The solution was incubated at room temperature on a shaker for 2 hours, and the TuBB-9-FITC conjugates were purified with a NAP-5 Sephadex column (GE Healthcare). After elution with TBS (10 mM Tris-HCl at pH 8.2, 150 mM NaCl), TuBB-9-FITC was concentrated with Microcon filter tubes (Millipore, USA) and resuspended in TBS (pH 7.4). In order to determine the labeling efficiency of FITC dye molecules per antibody the antibody concentration C_prot and FITC molecule number η were estimated by spectral measurements. For calculation the absorption at 280 nm and 495 nm was used in the empirical equations (1) and (2).


Calculation from absorption measurements showed, that between 1.5 and 2.4 molecules of FITC were bound to each antibody.