Immunoaffinity Gel Generation

1. Weigh 3 g of freeze-dried CNBr-activated Sepharose 4B powder into a 50-mL centrifuge tube; add 20 mL of 1 mM HCl (pH 2.0-3.0) to suspend the gel. Three grams of freeze-dried powder will swell to approx 10 mL of gel and the scale of the preparation may be altered to suit the application. Pour the gel onto a sintered glass filter (porosity C) and wash carefully with 600 mL (200 mL per gram of gel) of 1 mM HCl over a 15-min period. The washing solution should be added in several aliquots and great care should be taken to prevent the gel from drying. If necessary, flow through the funnel may be increased by using a slight vacuum, but it is important to wash slowly to properly prepare the gel.

2. Mix 50 mg of IgG (see Note 2), dissolved in 20 mL of 0.1 M NaHCO3, pH 8.3/0.5 M NaCl, with 10 mL of gel (Subheading 3.1., step 1) and divide into two 50-mL centrifuge tubes (see Note 3). Rotate the slurry with end-to-end tube rotator (for example, Roto-Torque Heavy Duty Rotator Model 7637-01, Cole-Parmer Instrument Company, Vernon Hills, IL) at 4°C for approx 14-16 h or 2 h at room temperature.

3. Centrifuge the gel at 200g for 5 min. Combine the supernatant from each tube and retain for measurement of unreacted antibody.

4. Wash away unbound IgG with coupling buffer (0.1 M NaHCO3, pH 8.3/0.5 M NaCl). Save the wash supernatants for the determination of unreacted antibody. The coupling buffer wash solution should be at least five times the gel volume.

5. Mix the gel with 10 mL of 0.1 M Tris-HCl, pH 8.0, per tube to block remaining unreacted groups on the cyanogen bromide activated gel. Using an end-to-end rotator, allow the gel to react for 2 h at room temperature. The Tris-HCl buffer contains primary amino groups that will react with any remaining active groups on the gel.

6. Wash the gel with 0.1 M acetate buffer pH 4.0/ 0.5 M NaCl followed by 0.1 M Tris-HCl, pH 8.0/0.5 M NaCl. Repeat twice more for a total of three cycles of the two-buffer wash. The washing solution should be at least five times the gel volume for each cycle.

7. Store the finished product at 4°C in PBS, pH 7.2, 0.02% sodium azide until used.

8. Determine the efficiency of IgG immobilization to the Sepharose beads by measuring protein concentration in the supernatants obtained from steps 3. The protein remaining in these supernatant fractions represents antibody that was not immobilized. The efficiency of conjugation may be calculated by:

Percentage Bound = 100 [(total mg IgG added) - (mg IgG in superna-tants)]/(total mg IgG added)

Efficiencies less than 70% indicate that inefficient IgG immobilization has occurred. The cause of inefficient coupling is often the presence of a buffer component (free amines in Tris-HCl buffers, for example) that competes with primary amines of the antibody for binding at the active sites of the cyanogen bromide activated Sepharose beads.

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