Physico-chemical separation

Ammonium Sulphate Precipitation

Because of their higher hydrophobicity, antibodies precipitate at lower concentrations of ammonium sulphate than most proteins and other serum components. Ammonium sulphate precipitation is then frequently used to enrich and concentrate antibodies from serum, ascitic fluid or cell culture supernatant.

The selectivity, yield, purity and reproducibility of precipitation depends upon several factors, including time, temperature, pH and rate of salt addition. Ammonium sulphate precipitation provides sufficient purification for some antibody applications, but most often it is performed as a preliminary step before column chromatography or other purification method.

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Size Exclusion

Dialysis membranes, size exclusion resins, and diafiltration devices that feature average molecular weight cut-offs can be used to separate immunoglobulins (>140kDa) from small proteins and peptides. However, except with specialised columns and equipment, these techniques alone can not purify antibodies from other proteins and macromolecules with similar molecular weight present in typical antibody samples. Gel filtration and dialysis are more commonly used following other purification steps, such as ammonium sulphate precipitation.

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Ion Exchange Chromatography

Ion exchange chromatography (IEC) uses positively or negatively charged resins to bind proteins based on their net surface charge in a buffer with a given pH, according to their isoelectric point (pI). Particularly in commercial operations involving production of monoclonal antibodies, conditions of target antibody binding and release can be determined with a high degree of precision, and therefore allow to purify antibodies and proteins with high resolution. Once optimised, IEC is a cost-effective, gentle and reliable method of antibody purification.

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Purification of Antibodies using physicochemical separation

Class M immunoglobulins

Class M immunoglobulins (IgM) form a pentameric structure through attachment via the Fc region of the five base units. Because of the steric hindrance around the Fc regions, protein A and protein G bind IgM very weakly. On the contrary, as the light chains are more easily accessible, protein L can be used to purify IgM provided that the light chains subtype is suitable. Nevertheless, IgG exhibiting the same light chain subtype present in the sample will also be purified.

To circumvent this problem, IgM are usually purified by a combination of physico-chemical separation techniques, including ammonium sulphate precipitation, followed when appropriate by ion exchange chromatography. Another way of purifying IgM consists in an ammonium sulphate precipitation followed by removal of IgG with protein A or G.

After ammonium sulphate precipitation, purified IgM are delivered in a phosphate buffer (KH2PO4 and K2HPO4 to 150 mM, pH 8, NaCl 1 M) containing 0.02% sodium azide. If you do not wish azide to be added, please inform us while placing your order. Antibody purity is controlled by gel electrophoresis.

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Class Y immunoglobulins

IgY are a particular class of immunoglobulins produced mainly by birds and reptiles. They have the same function as mammalian IgG but their respective Fc regions differ significantly (see the Host section). Therefore protein A and G can not be used to purify IgY. These immunoglobulins are usually purified from egg yolk by selective precipitation, followed when appropriate by ion-exchange chromatography. If necessary, egg yolks can be separated and stored frozen for subsequent extraction of antibodies.

IgY produced by chicken are naturally present at high concentrations in egg yolks. As a general rule, a single egg yolk from an immunised chicken contains approximately 300 mg of IgY.

Following selective precipitation, purified IgY are resuspended in PBS containing 0.02% sodium azide. If you do not wish azide to be added, please inform us while placing your order.

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