Kris Cahyo Mulyatno ITD UA

AFFINITY PURIFICATION OF ANTIBODIES

(Fisher et al.,1988, Cell 54: 813-822) Conjugation of carrier (BSA) to peptide 1. Dissolve pure BSA @ 20mg/ml in 10mM phosphate, pH 7.0. 2. Dissolve MBS-SMCC [m-maleimidobenzoyl N-hydroxysuccinimide ester succinimidyl 4- (Nmaleimidomethyl)cyclohexane-1-carboxylate] @ 25mg/ml in NNdimethylformamide. 3. Mix 20ml SMCC and 200ml BSA (500mg:4000mg or 1:8); incubate ON @ RT. 4. Separate conjugate from unreacted crosslinker on a G25 column in 100mM phospate, pH 6.2. Identify the peak by A280 and pool peak (1 ml) fractions. 5. Dissolve peptide (1-5 mg)in 1ml 0.1M Borate, pH 9.1. Reduce by adding 100ml 0.1M NaBorohydride (NaBH4), mix. Let stand 10'. Lower pH with 70ml 1M HCl to inactivate NaBH4. Neutralize with 40ml 1N NaOH. 6. Combine carrier and peptide and mix gently ON. 7. Fractionate over G25 ON in 0.1X PBS, A280, pool peak and lyophilize. 8. Check peptide coupling (1mg samples) on a Laemmli gel. Affinity Column Preparation 1. Affigel 102 (BIORAD) washed in PBS on a scintered glass funnel (3ml/column). 2. Transfer to 15ml screw cap tube in minimal volume. 3. Add 7mg SMCC (in DMF) to 2.5ml beads and mix for 2h @ RT. 4. Wash beads again in PBS. 5. Resuspend in minimal volume 100mM phosphate, pH 6.2. 6. Add BSA-peptide and react ON with mixing. 7. Wash beads extensively. 8. Pack into a 3ml syringe as column. Add azide! Affinity Purification 1. Equilibrate column with PBS, pH 7.4. 2. Load on sample diluted (1:10 ?)to run slowly ON (e.g., 160ml @ 8ml/h = 20h). 3. Rinse column: 50ml 2M NaCl, pH 7.5 50ml 0.1M Borate, pH 9.1 50ml 0.1M PBS, pH 4.5 4. Elute antibody with 20mM glycine-HCl/200mM NaCl. Collect 0.8ml fractions into tubes containing 0.4ml 0.1M Tris-HCl, pH 8.5. 5. Assay fractions by A280 and pool peak. Store some @ 4oC (+0.1% NaN3) and freeze remainder @ -70oC.