Streptavidin coated magnetic beads

Cells grown to subconfluency were labelled with EZ-link Sulfo-NHS-LC-Biotin (Pierce Biotechnology,) according to manufacturer’s instructions but adapted to adherent cells. Briefly, the cells were washed trice with cold PBS, labelled for 30 minutes at RT with 2 mM biotin. Labelling was quenched with 1 mM cold Tris-HCl, pH 7.4, washed once with Tris-HCl and twice with cold PBS. The cells were scraped in cold lysis buffer (20 mM Tris-HCl pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% NP40, 10% glycerol) on ice and then lysed on ice for an additional 30 minutes.
Biotin-labelled lysate was coupled to 20 µl streptavidin coated magnetic beads (Roche Diagnostics) on a rotating wheel for 30 minutes at RT. Unbound lysate was removed using a magnetic concentrator and the beads washed twice with lysis buffer and once with M-PBS. Tomlinson I+J libraries were added in M-PBS and incubated on the rotating wheel for 1½–2 hrs at RT. Beads were washed 15 times with PBS-T and 15 times with PBS with 2 minutes incubation each time. After washing, the remaining phages were eluted by adding 100 μl trypsin in PBS (1 mg/ml). After 10 minutes the eluent was infected into TG-1 (OD₆₀₀ 0.5) and spread on TYE plates with 100 μg/ml Ampicillin and 1% (W/V) Glucose. Plates were incubated at 30 °C overnight.

Each polyamine at 10 mM concentration was prepared in PBS, pH adjusted with HCl to pH 8.0. EZ-Link NHS-LC-Biotin (Thermo Scientific) at concentration of 20 mM was prepared in DMSO. The reaction containing 0.5 ml of each polyamine, 0.3 ml NHS-LC-biotin and 0.2 ml PBS was incubated for 1 h at room temperature. The reaction was stopped by the addition of 1 ml 10 mM ethanolamine in PBS and incubation for 1 h at room temperature.
To immobilize biotinylated polyamines on streptavidin-coated magnetic beads (Roche), 50 μl (10 mg ml⁻¹) magnetic beads were washed with 100 μl PBS for three times and then were incubated with 0.2 mM biotinylated polyamines (10 nmole) in 50 μl PBS for 1 h at 4 °C, followed by washing with 100 μl PBS for three times and resuspended in 50 μl PBS. After removing the supernatant, 10 μl magnetic beads containing biotinylated polyamines were incubated with 3 μg RAD51 in 20 μl reaction buffer (35 mM Tris-HCl, 1 mM DTT, 1 mM ATP, 0.25 mM MgCl₂, 35 mM KCl) for 20 min at 37 °C. The streptavidin-coated magnetic beads were captured and then treated with 20 μl of 2% SDS to elute any RAD51 that might have been retained on the beads. The supernatant and eluate, 9 μl each, were analyzed by 10% SDS–PAGE and Coomassie Blue staining.

All the experiments were performed using nuclear extracts from cultured HeLa non-confluent cells (Ipracell S.A., Belgium). Benzonase was from Merck and DNase I was from Invitrogen. The magnetic streptavidin-coated beads were from Roche. The pUC 19 plasmid (Invitrogen) was used as a template for PCR-based production of biotinylated duplex DNA oligonucleotides. The PCR primers endowed with a photocleavable biotin moiety were from Eurogentec. Colloidal G250 Coomassie blue was from Thermoscientific.