Pc04n

Low electrophoretic mobility avidin-labeled beads were made from 1.5 µm diameter carboxypolystyrene beads (PC04N, Bangs Laboratories, Fishers, IN). Fifty µL of beads were washed 2× in 200 mM 2-(N-morpholino)ethanesulfonic acid (MES, pH 6) with 0.1% sodium dodecyl sulfate (SDS) and resuspended in 500 µL of MES buffer and mixed with 500 µL of 10 mg/ml EDC (Sigma-Aldrich, St. Louis, MO) to activate the carboxy groups. After 15 minutes, beads were washed with 200 mM sodium phosphate buffer (pH 7.5) with 0.1% SDS. The washed beads were resuspended in 150 µL and reacted with 1 µM avidin (Sigma-Aldrich, St. Louis, MO) and 25 mM 1 kDa amino-PEG (#26116, Pierce, Rockford, IL) for 3 hours at room temperature. Beads were washed and resuspended in 10 mM Tris (pH 8) with 50 mM NaCl and stored at 4 °C. Where indicated, 1 µm diameter streptavidin coated beads (CP01N, Bangs Laboratories, Fischers, IN) having high electrophoretic mobility were used in place of the custom beads.