Updated by DAS 6/97.

This protocol requires that an appropriate affinity column is prepared, using purified protein from native sources or from bacterial expression. Suitable matrices are Sepharose or Affi-gel which are activated to covalently bind your protein to the resin.

1. Wash the column (~1 ml resin bed) to remove residual protein before each use using 10 column volumes of the following sequence of buffers:

0.2 M glycine, pH 2.8 ~10 ml

0.1 M NaHCO₃, pH 8.5, 0.5 M NaCl ~10 ml

Repeat the cycle with the above buffers twice. Then equilibrate the column in TTBS buffer (0.3 M NaCl, 20 mM Tris/Cl, pH 7.8, 0.1% (v/v) Tween-20 and 0.01% NaN₃) containing NaCl adjusted to 0.5 M. {To make this, add 25 ml 4 M NaCl to final volume of 500 ml TTBS.}

2. Prepare serum. Thaw one 10 ml aliquot of serum, add 1 ml 10X TTBS and 0.55 ml 4 M NaCl to the serum. Centrifuge the serum to remove any precipitate.

3. Absorb the antibody batchwise by transferring the affinity resin to the tube (a 15 ml tube) with the serum. Incubate on a rotator ON in the cold room. (I do the batch method exclusively now). Alternatively, you can apply the serum to the column using a slow flow rate. I do these procedures at my bench but keep buffers and eluted fractions cold in an ice bucket. Since the volumes are low, I manually collect fractions and do not set up a siphon delivery system. Collect the serum that passes through the column and re-apply it a second time using a slow flow rate. Save the serum after the final application in case something went wrong or all the antibody is not recovered by the first absorption.

4. Wash the column extensively with TTBS+NaCl to 0.5 M until the A₂₈₀ is less than 0.02. Collect 10 ml fractions of the washes and check the A₂₈₀ of the fractions. Drain the column to the top of the resin bed but don't dry out the resin.

5. Elute the antibody using 0.2 M glycine, pH 2.8, containing 0.02% NaN₃. Elute using 1 ml aliquots of buffer. Collect fractions into 1.5 ml microfuge tube containing 50 ml 1 M Tris, pH 8.5. This neutralizes the acidic elution buffer soon after the protein is eluted. Collect at least 10 fractions. This is usually sufficient to remove the antibody. Read the A₂₈₀ of each fraction using an appropriate blank (i.e., 1 ml glycine buffer plus 50 ml 1 M Tris).

6. Pool the appropriate fractions. I usually make 2 pools--one that has the most concentrated protein in it (usually 2-3 fractions) and a pool of all the remaining protein-containing fractions. Get an A₂₈₀ of the pools. Dialyze pool against PBS+azide ON in the cold room. Store antibodies at 4°C or consider freezing (-70 °C) in aliquots in the presence of 50% glycerol.

7. Wash the column by washing extensively with the 0.2 M glycine, pH 2.8 buffer, followed by TTBS. Store it at 4°C.