3.3.1. Preparation of Peptide-Agarose
1. Mix 1.5 g of CNBr-Sepharose and 200 mL of 1 ml HCl and leave for 15 min at room temperature.
2. Collect the slurry on a sinter funnel, drain until a moist cake is formed. Add the cake (approx 5 mL volume) to 5 mL of PBS (pH 7.5-8.0) containing approx 500 ^g of peptide. Agitate gently for about 2 h at room temperature. Note: Do not use a magnetic stirrer as this fragments the resin and generates fines that may slow or block the column flow.
3. Pour the slurry onto a sinter, wash sequentially with 20 mL of the following: PBS, pH 7.2; 100 ml sodium acetate, pH 4.0; 2 M NaCl in PBS; TBS, pH 8.0. Store as a 50% slurry in TBS containing 0.1% sodium azide at 4°C.
3.3.2. Preparation of Serum
1. Allow clot to form then ease away from sides of tube (to prevent clot adhering to the sides) and leave overnight at 4°C.
2. Aspirate the supernatant (serum) and clarify by centrifugation at 1000g for 5 min.
3. Add 5 mM ethylene diamine tetraacetic acid to the sample then add 0.82 vol of saturated ammonium sulfate solution while stirring and leave for 15 min at room temperature.
4. Collect the pellet by centrifugation (10 min, 10,000g, 4°C).
5. Redissolve the pellet in its original volume using TBS. Add 10% NP40 to a final concentration of 0.1% and spin in a microfuge to clarify.
1. Use an APBiotech reversible column. Pack 2 mL of affinity matrix into the column (keep moist) in running buffer (TBS containing 0.1% NP40). Wash with 20 mL of running buffer over 20 min.
2. Run in the antibody solution as prepared earlier. The flow-rate should be approx 1-2 mL/min (a peristaltic pump is useful to control the flow-rate). Run in the equivalent of about 1 mL of antiserum per mL of gel.
4. Reverse the direction of flow through the column and wash with 10-column volume of TBS containing 0.1% NP40 for 10 min; five-column volume of TBS at the same flow-rate; and five-column volume of 0.9% NaCl.
5. Elution of antibody may be achieved by either one of the following two procedures.
a. Low pH elution:
1. Elute the bound antibody with four-column volume of 100 ml sodium citrate, pH 2.5, for 10 min.
2. Collect the eluate and immediately neutralizing to pH 5.0-8.0 with 2 M Tris-HCl base. This can be achieved by prealiquoting into the collecting tubes the amount of Tris base necessary to neutral ize a given fraction volume.
3. Assay fractions by ELISA (see Subheading 3.2.) diluted 1 in 5 to 1 in 50.
4. Pool the most strongly positive fractions and adjust the pooled fractions to pH 6.0.
5. Add 1 vol of saturated ammonium sulfate solution.
6. Leave for 10 min at room temperature and pellet the antibody at 10,000g for 10 min at 4°C.
7. Resuspend the antibody in water at approx 1-5 mg/mL (OD280 of IgG is about 1.4 for a 1 mg/mL solution). Either: dialyze against TBS containing 0.1% sodium azide or add one-tenth volume 10X TBS (see Note 10). Store in aliquots at -20°C.
b. Elution with 4 M MgCl2:
1. Elute the bound antibody with four-column volumes of 4 M MgCl2.
2. Dilute the eluate 10X with distilled water.
3. Add an equal volume of saturated ammonium sulfate and pellet the immunoglobulin at 10,000g.
4. Resuspend the pellet in water.
5. Either: dialyze against TBS containing 0.1% sodium azide or add one-tenth volume 10X TBS (see Note 10). Store in aliquots at -20°C.
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