PREPARATION OF PROTEIN A SEPHAROSE CL 4B BEADS

Swelling and Storage

1.Resuspend .2 gm of beads (= 1.0 ml swelled bead volume)in 40 ml of distilled water.Let swell for at least 2 hours.

2.Wash beads twice in (10 ml each wash).Spin in clinical or table top fuge.Do not exceed 2K rpm.

3.Resuspend in Storage buffer: T.E.+ 0.1% Azide

4.Make 50% slurry with storage buffer.Add enough storage buffer so that final volume of beads plus storage buffer is twice the volume of beads alone (eg.If sedimented volume of beads is 1 ml,then add enough storage buffer so that the total volume of beads plus buffer is 2 ml).

Blocking and Use

1.Remove desired volume of 50% slurry (30 µl of slurry per I.P.or Chip).Put in 15 ml conical tube.

2.Spin in table top

3.Resuspend beads in cold blocking buffer:

4.Mix Overnight in cold room (couple of hours is probably fine)

5.Wash once with 10 ml cold blocking buffer

6.Make 50% slurry with blocking buffer.Add enough blocking buffer so that final volume of beads plus blocking buffer is twice the volume of beads alone (eg.If sedimented volume of beads is 1 ml,then add enough blocking buffer so that the total volume of beads plus buffer is 2 ml).