Each week, our lab staff at Novus Biologicals produce, conjugate and purify hundreds of products for our antibody catalogue. Purification protocols are specific to each antibody. For example, we recently purified rabbit polyclonal anti-OCT4 to a final concentration of 1.2 mg/m, using a peptide affinity column. We also purified 20ml of c-Myc ascites over a protein G column, to create purified mouse mouse monoclonal anti-c-Myc (9E10) antibody to a final concentration of 0.82mg/ml.
Improved immunoassay technology means antibodies must be extremely pure, with minimum risk of false positives and “noisy” results. A wide range of purification methods exist to ensure this is possible. Which one we use depends on the Ab class, the species it was raised in and its intended use.
Antibodies are prepared from serum, tissue culture supernatant or ascites (peritoneal fluid containing immunoglobulins) and then purified by one or more of the following methods: Protein A/G purification; Gel filtration; Ammonium sulphate precipitation; Ion exchange chromatography and immunoaffinity purification. The final product is supplied with a full protocol sheet and QC report stating yield, purity and immunoreactivity analysis.
Affinity column immunopurification is commonly used to produce antipeptide antibodies. The peptide is first immobilised on sepharose beads, and the serum is then loaded onto an affinity column. The bound antibodies are eluted, and ELISA is performed on both the immunopurified Abs and the crude serum.
Antibody purification on a protein column (A or G) is similar, but following ELISA the proteins undergo further SDS-PAGE analysis. This yields 2 – 4 mg/ml of serum. Another purification method is IgY purification from egg yolk extract, on an ion exchange column.
