4.2. Preparation of Anti-FLAG agarose beads

We recommend using Anti-FLAG agarose beads from Sigma (Cat # A2220) for the purification of L1-RNPs. The Anti-FLAG agarose beads are supplied in 50% glycerol with buffer. Thoroughly resuspend the resin by gently inverting the container several times.

Transfer 40 μl Anti FLAG agarose gel suspension using a blunt cut 200 μl tip into a 1.5 ml tube. (40 μl bead suspension is equivalent to 20 μl packed gel beads which will efficiently bind ~2 μg FLAG-tagged protein).

Add 1 ml cold beads in wash buffer [100mM KCl, 5mM MgCl₂, 10 mM HEPES (pH-7.0)], mix with resin by inverting the tube several times and then centrifuge at 2000 rpm for 1 min at room temperature (RT). Keep the tube on ice for 2 minutes to settle the beads at the bottom of the tube. Remove the wash buffer without disturbing the resin. Repeat the washing steps twice more.