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120 Casey et al.
3) Mix the washed medium and the peptide in a 15-ml tube, adjust the pH to 6–8, and
the volume should be made to 2 ml with coupling buffer. The coupling reaction
should be allowed to proceed overnight at 4ºC, mixing very slowly end-over end
on a rotator.
4) After the coupling is completed, excess ligand should be washed away with 10
CV of coupling buffer, and any non-reacted groups on the medium should be
blocked by mixing and standing in 1 M Tris–HCl buffer, pH 8, for 2 h.
5) To wash the medium after coupling, four alternative washes with high and low
pH should be used. Each cycle consists of a wash with 10 ml of 0.1 M Tris, pH 8,
containing 0.5 M NaCl, followed by 10 ml of 0.1 M Na-Acetate, pH 4, containing
0.5 M NaCl.
6) The coupled affinity resin should be resuspended in PBS, transferred to an empty
column and washed with 20 ml PBS. The column should be stored at 4ºC; for
long-term storage, the column should be stored in 20% ethanol.
3.9. Affinity Purification Using Peptide Column (See Note 3)
In our example, the peptide column is used to purify antibodies having an
affinity for the peptide mimotope from human serum. Ethical approval should
be obtained for use of human serum. Collect all fractions.
1) Filter human serum (2 ml) using a 0.2-μm filter and dilute 1:10 in PBS. Retain a
small sample for analysis.
2) Equilibrate the 2-ml peptide column with 10 CV PBS.
3) Load diluted serum onto the column, taking care not to disturb the resin, and
allow to pass through the column slowly. Note the flow as it may be stopped at
any time, and this allows longer contact time for the serum antibodies with the
peptide resin.
4) Repeat step 3 passing the serum through the column again and collecting the flow
through. Steps 3 and 4 should take at least 1htoensure sufficient contact time
of the serum antibodies with the peptide resin.
5) Wash the column with 50 ml PBS.
6) Wash the column with 50 ml wash buffer.
7) An additional 50 ml PBS wash should be carried out to ensure all the non-specific
serum components are washed away.
8) To elute the bound antibodies, 10 ml of elution buffer is added and 10 × 1 ml
fractions collected. Fractions are immediately equilibrated with 2 M Tris and
stored at 4ºC prior to analysis.
9) The column is re-equilibrated with 50 ml PBS and stored at 4ºC.
3.10. Analysis of Affinity-Purified Antibodies to Ensure
Validity of Column
It is important to assess the efficiency of the peptide affinity resin and identify
which of the eluted fractions to pool. Sodium dodecyl sulfate–polyacrylamide