PREPARATION OF PROTEIN A SEPHAROSE CL 4B BEADS
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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:
| T. E. | 25 ml of T.E |
| 0.1% Azide | 25 µl of 10% soludtion |
| 0.1% BSA | 25 mg of BSA (Fraction V, powder) |
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).
