The generation of polyclonal antibodies to an antigen of interest is an important technique applicable to many areas of biological research. Seattle Genova provides custom production of Drosophila antigen specific polyclonal antibodies. Our antibody production capabilities include immunogen preparation, rabbit immunization, serum titer testing, immunoaffinity purification. Further polishing purification services offered include size exclusion chromatography, buffer exchange by dialysis, endotoxin testing, analytical SEC, and SDS-PAGE.
Drosophila melanogaster is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. D. melanogaster is typically used in research owing to its rapid life cycle, relatively simple genetics with only four pairs of chromosomes, and large number of offspring per generation. About 75% of known human disease genes have a recognizable match in the genome of fruit flies, and 50% of fly protein sequences have mammalian homologs. Drosophila is being used as a genetic model for several human diseases including the neurodegenerative disorders Parkinson's, Huntington's, spinocerebellar ataxia and Alzheimer's disease. The fly is also being used to study mechanisms underlying aging and oxidative stress, immunity, diabetes, and cancer, as well as drug abuse.
Despite the typical immunization and tittering process, we are offering two protocols on the rabbit polyclonal antibody purification part.
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.
Polyclonal antibodies can be produced quickly and relatively cheaply and do not require the same amount of expertise or time as monoclonal antibody production. They can also be very specific and high concentrations can be purified from relatively small amounts of serum. However, polyclonal antiserum contains a heterogeneous population of antibodies, which can be hard to reproduce in subsequent immunizations. Unlike monoclonal antibody production, a consistent source of antibodies cannot be generated.
Antigen Affinity Purification of Antibodies
Although Proteins A, G, A/G and L are excellent ligands for purification of total IgG from a sample, purification of antigen-specific antibodies is often required. This can be performed by immobilising the antigen of interest (e.g., the peptide used to raise the antibody) on a solid phase so that the antibodies that bind specifically to the antigen are retained upon loading of the serum, while other impurities and unspecific IgG are discarded in the flow-through.
Our protocol provides many advantages such as very high purity levels (>99% can be achieved in one step), very high selectivity, and therefore very high resolution, including certitude that the antibody is specific for the antigen of interest. The polyabs are purified using a specific 3-step antigen affinity purification procedure, for those antibodies require an extremely specific separation between antibodies which are specific for the control peptide and those which are specific to the modified peptide.
This method has proved useful across a variety of types of antigens, including peptides, and for purification of even epitope-specific antibodies.
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