Koshland Web Site/Methods
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:

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).

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