Bio-Rad CM Affi-Gel Blue Gel - Manual (Page 5)

Instructions for a Protease Free
Globulin Fraction From Serum
1. Follow the general instructions through step 5.
2. Measure the total volume of the collected protein peak.
Determine the amount of solid ammonium sulfate required
to achieve a 45% saturated solution.
*** While stirring the
pooled protein with a stir bar on a stir plate, slowly add the
ammonium sulfate to the solution.
3. After all the (NH
4
)
2
SO
4
has been added, continue stirring
for at least 1 hour at room temperature. If stirred or stored
overnight before centrifugation, the suspension should be
kept at 4 °C.
4. Centrifuge the suspension in a refrigerated (4 °C) centrifuge
at approximately 1,000 g for 20 minutes. (If supernatant does
not appear clear, centrifuge longer.) Discard supernatant.
5. Resuspend the pellet in 45% saturated ammonium sulfate
by breaking up pellet with a pipet and centrifuge again.
6. Resuspend pellets in a minimum volume of running buffer.
Do not mix vigorously to dissolve pellets (this will denature
proteins). If pellet does not go into solution readily, let sit in
PBS at 4 °C for 1–2 hours, and then mix.
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7. Remove any remaining ammonium sulfate, by desalting on
Bio-Gel P-6DG desalting gel or prepacked desalting
columns using gravity flow, with the Econo-Pac
® P6 car-
tridge, or by dialysis.
8. Continue with step 6 of the general instructions for CM
Affi-Gel blue gel column regeneration.
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