Antibody Purification Options
The following is a list of our current antibody purification services. If you are in need of special purification, large quantities or you do not see a package that suits your needs, please feel free to contact us for a quote.
Antibody Purification Methods
Antibody purification can be divided into two groups: precipitation methods and chromatographic methods. The latter is grouped further into non-affinity and affinity chromatography.
The choice of purification methods available include:
- Polyethylene Glycol (PEG) and /or Ammonium Sulphate Precipitation
- Immunoaffinity purification
- Protein A affinity Chromatography
- Protein G Affinity Chromatography
- Protein A/G affinity chromatography
- Ig Antibody Affinity Purification
- Antibody Purification by Ion exchange chromatography
- Hydrophobic interaction chromatography
- Size exclusion chromatography
- Ammonium sulfate precipitation
- HPLC purification
- Procedures and methodology used for the purification and Quality Control report (including analysis of yield, purity and immunoreactivity)
are provided with each purified product
Antibody purification using Protein A, G, or A/G
Protein A/G is a recombinant or genetically engineered protein that combines both Protein A and Protein G binding properties. Protein A/G is designed to contain four Fc binding domains from Protein A and two from Protein G. Protein A or Protein G purification removes the IgG fraction based on the specificity of these proteins for the Fc portion of the IgG. Protein A is produced from Staphylococcus aureus. It has the capacity to bind at least two molecules of IgG. The binding is specific to the Fc portion and does not affect the antigen binding sites. Protein G is isolated from Group G streptococci and binds the Fc region of the IgG in a similar manner to Protein A. Protein A and Protein G have differing binding efficiencies for IgG from different species. It is important to check this before deciding which method to use. For example, Protein G works well with sheep Ig, but Protein A does not. Neither Protein A nor Protein G will bind to chicken Ig. Care should be taken when eluting the antibody from the column to avoid denaturation of the antibody.
Peptide Immunoaffinity Purification
The most commonly used method to purify antigen-specific antibodies from crude sera is immunoaffinity purification. Unlike Protein A or G, the non-specific Ig fraction is not retained. In this procedure, peptide antigen is bound covalently to a solid support. The antibodies within the polyclonal sample that are specific for the peptide antigen bind to the support column. The unbound antibodies are removed from the column by washing and the specific antibodies are eluted from the column. The product of immunoaffinity purification is highly specific antibodies. Immunoaffinity purification can occasionally cause denaturation of the antibody due to the conditions used to elute the bound antibody from the column. It is important to compare the response generated by the purified sample against the response generated by the crude sera.
IgY Purification from Egg Yolk
Chicken antibodies IgY are the equivalent to mammalian IgG antibodies and can be isolated from serum or egg yolk. The egg yolks contain at least the same concentration of IgY antibodies found in chicken or rabbit serum. IgY can be used in most biochemical and cellular applications, including Western Blots. As the Fc region of chicken IgY is sufficiently different from mammalian IgG chicken antibodies do not cross-react with mammalian proteins, and do not bind with mammalian rheumatoid factors, Fc-receptors or proteins A or G. Therefore, the background signal or false response in certain immunochemical assays is reduced.
Polyethylene Glycol and /or Ammonium Sulphate Precipitation
This method is useful for concentration and partial purification of antibodies from most sources and all species including purification of IgY's from chicken egg yolks. Although on its own the yields of antibodies are impure, it is the method of choice when combined with ion exchange for large volumes of antisera and or followed by Antigen Affinity purification to enrich for specific antibodies. It is not recommended for purification of tissue culture supernatant.
Ig Antibody Affinity Purification
This method can be used to isolate monoclonal antibodies from supernatants which contain high levels of serum Ig. It is the choice for secondary antibody production.
Size Exclusion/Gel Filtration Chromatography
A single gel matrix or a sequence of size exclusion columns can be used for purification of IgM and IgG antibodies. The purification strategy employed is dependent on the immunoglobulin class and the source of the material.
Antibody Purification by Ion Exchange Chromatography
This is usually the method of choice when purifying antibodies from ascites, as the monoclonal can be resolved from the host immunoglobulins. It has been used in adjunct to ammonium sulfate precipitation for purifying antibodies from ascites fluid or where Protein A and Protein G elution conditions may damage the antibody. It is also applicable to all IgG isotypes.
Aseptic/Low Endotoxin Removal, Purification and Testing
Purification under aseptic conditions in a dedicated, limited access facility to generate clean, low-endotoxin antibody preparations.