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A 15-ml starting serum sample may have to be passed over this column several times to quantitatively deplete cross-reactivity. The serum may be analyzed after each or several passes, or an aliquot can be saved for analysis at a later time.

4. If cross-reactivity with homologous phosphoprotein(s) is anticipated, pass the flow-through through the homologous phosphopeptide affinity matrix column(s) using the methodology described in step 3.

Refer to Strategic Planning for discussion of cross-reactivity with homologous phospho-proteins.

Purify antibodies

5. To purify antibodies by positive-selection affinity chromatography and dialysis, hydrate and wash ~25-cm strips of dialysis tubing in advance for collection of fractions from the positive-selection affinity purification. Secure one end of each length of tubing with a dialysis clamp and check for leaks (unit4.4). Also prepare 6 liters PBS/azide and cool to 4°C in advance for use as the dialysis solution.

To minimize the time that the antibodies are exposed to the elution solution, fractions (~3-ml each) from the positive-selection affinity column should be collected directly into prepared dialysis tubing.

6. Pass the flow-through from the previous chromatography step through the positive-selection phosphopeptide affinity column three times, without washing between passes.

Three passes are performed to maximize the interaction of the antibody with the affinity matrix.

7. Collect the flow-through from the final pass, then wash the column with 5 to 20 ml PBS/azide (depending upon the pre-column volume and column bed volume) and combine the washings with the flow-through. Wash the column with an additional 20 ml PBS/azide, and collect the washings separately as the "wash" fraction.

8. Elute with chaotropic agent of choice: either 20 ml of 3 M NaSCN, or 10 ml of 3.5 M MgCl2, followed by 10 ml of 4.5 M MgCl2. Upon starting the elution, immediately begin collecting ~3-ml fractions directly into the dialysis bags prepared in step 5, securing the proximal end of the tubing with dialysis clamps and dropping the bag immediately into the stirring 4°C PBS/azide dialysis solution. Collect at least six fractions. This elution step can be repeated one to three times for better recovery.

Because antibodies are eluted from the positive-selection affinity column in strongly chaotropic solutions that are potentially deleterious to the stability of the antibody, it is highly recommended to collect the fractions in prepared dialysis tubing so that they may be immediately placed into the PBS dialysate, thus, minimizing the time that the antibodies are exposed to the eluting solutions. Although MgCl2 is thought to be "gentler," the authors have found that gravity-driven flow rates from phosphopeptide columns eluted with this salt quickly become extremely slow, possibly because of precipitation of the salt in the column or an interaction of the Mg2+ ion with the phosphate groups. Thus, use of NaSCN is preferred, and this salt appears to permit recovery of comparable activity.

9. Dialyze all fractions collected in step 8 exhaustively against PBS/azide at 4°C

Analyze and store purified antibody

10. Determine protein concentration of dialyzed fractions by measuring absorbance at 280 nm or by a colorimetric protein assay (unit3.4) and calculate yield.

Generally, >1 mg of purified antibody is recovered from a 15-ml serum sample.

11. Store the antibody in aliquots at -20°C or colder for long term storage; store working aliquots at 4°C with 0.05% sodium azide to prevent bacterial growth.

Post-Translational Modifications: Phosphorylation and Phosphates

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