Surface preparation
for the bond-force measurements

For the bond-force measurements of the ligand-receptor bonds, the surface of the samples need a special preparation. This was done by PAUL-BERTRAM KAMP within a collaboration of the Sonderforschungsbereich 613 (Project K3). The surface preparation was always done after the following recipe:

Before any biological preparation of the surface, the sample is heated at 120 for 5min to dry it. The surface is then silanized with an epoxy-silane by heating the sample for two hours at 80 in a solution of 2.5% 3-Glycidyloxy-propyl-trimethoxysilane in xylol and 0.1% triethylamin (all chemicals from SIGMA-ALDRICH, Taufkirchen, Germany). The silanisation is followed by 3 washing steps with acetone and finished by drying the sample at 100 for five minutes. On top of the silanized surface, 0.3$\mu$l biotinylated oligonucleotides (biotin-5'-agggttttcccagtcacgacgtt-3', SIGMA-ARK, Germany), diluted 1:1 with 58% DMSO, 40% Methanol and 2% TEMED, are pipette-spotted. Directly after spotting, the samples are stored in a damp atmosphere for 1hour. Then, the samples are heated for five minutes at 100 and exposed to 300mJ UV light. Three washing steps follow with a washing buffer of 50mM Na$_2$HPO$_4$, 10mM Tris-Base, 5mM EDTA, 0,1% SDS, 0.01% N-laurosyl sarcosine, 0.01% Tween 20, 0.01% Triton X100, 0.1M NaCl and 0.5% polyethylene glycol 4000 at a pH of 8.5 (adjusted with acetic acid) and another washing step with distilled water. To deactivate the excessive epoxide groups, the sample is incubated for 1 hour at 55 in 1M sodium acetate buffer containing 2.5% polyethylene glycol 4000 with a pH of 5.0. Finally, the samples are washed three times in washing buffer (see above) and one time in distilled water for five minutes. The samples are stored at room temperature until usage.

The concentration of the biotinylated oligonucleotides is a critical parameter for the experiments, since a too high concentration result in several bonds per bead and with a too low concentration, there are nearly no bonds at all. Both cases have to be avoided and, therefore, different concentrations were tested and also adjusted to the used beads (different beads have different binding properties). The final concentrations of oligonucleotide used in these experiments are between 10 and 1000nM.