Antibody Purification

Buffers:

Binding Buffer: 10 mM Tris pH=7.5

Salt Elution Buffer:

PBSS (phosphate buffered saline with 0.5 M salt):
Add 12.19 g of dibasic sodium phosphate (Na2HPO47H2O),
0.62 g monobasic sodium phosphate (NaH2PO41H2O)
and 37 g NaCl to 1 liter of water.
Simpler, use 5 instant PBS tablets (Sigma, P4417), and 29 g NaCl in 1 liter water.

Acid Elution Buffer: 0.2 M glycine pH=3.0

Columns:

There are a number of resins one can use to purify antibodies: a few proteins bind to general Fc portions of the Ig molecule and can be used to purify total Ig from serum or ascites fluid. Such molecules are e.g. Protein A, Protein G, Protein L. Other options are covalently linked immobilized target protein, which is then used to get a highly specific antibody prep. As all other proteins and antibodies should not bind to your target protein resin. Or anti-antibody antibody columns such as an anti-IgG column. Many such immobilized proteins are available through PIERCE

Purification:

Dilute or dialyze your crude solution into binding buffer, then apply slowly (1 ml / min flow; gravity flow; or batch bind) to your matrix. Wash the matrix with excess binding buffer (>20 column volume). Now elute with the salt elution buffer for 5-10 column volumes. Your antibody may not elute in this buffer in which case you have a more highly purified prep. eluting in the acid wash. Be sure to assay this fraction though. Next elute with 5 to 10 column volumes of acid elution buffer. Neutralize with 1:100 dilution of 1M Tris pH=10 or 1:10 dilution of 1M Tris pH=8. You should have five fractions to assay for activity by ELISA now:

Starting material, flow through, wash, salt elution, acid elution. Check each by ELISA to test for the presence and activity of your Ab. Dialyze each fraction against PBS and store as described. Columns can be regenerated with an acid wash at pH 2 in glycine or citrate, and should be stored in water containing 0.05% sodium azide.