applications and troubleshooting appendix chapter 21

Immunogen Design

Antigens Used for Antibody Production • You can use synthetic peptides, recombinant proteins, immunogens, and others. We do not recommend the use of whole cells for anti- body production. • Arbitrary selection of the antigenic peptide or use of a recombinant fusion protein usually will result in a 65-70% success rate using standard methodologies. If the antigenic peptide is chosen using standard bioinformatic algorithms, you can increase your success rate to 75-80%. If this optimized peptide is then conjugated using Invitrogen’s proprietary approach, you can increase your success rate to about 85-90%. A recent collaborative study showed that approximately 450 polyclonal peptide antibodies out of 500 individual protein targets recognized their target by western blot analysis.

Peptide Conjugation • Peptides are normally too short to be immunogenic. A carrier protein enhances immunogenicity. MAP and KLH are the two most popular conjugation methods. Other available conjugates are OVA (Ovalbumin), BSA (Bovine Serum Albumin), and THY (Thyroglobulin). • Conjugation is usually done through the sulfhydryl group of a Cys designed into the peptide. • For peptides < 6 kDa, conjugation will be required.

• If studying invertebrate species, you should use carrier proteins such as ovalbumin and KLH (from sea snail). KLH has no homology to 21.2 vertebrate proteins. Invitrogen utilizes a proprietary multiple conjugation method where we conjugate peptides through both termini and through key internal sites. This methodology presents the peptides in alternate, multiple conformations, making them more antigenic and producing antibodies appendix with greater utility across assays. Our conjugation scheme provides a better matching of epitopes between the presented peptide and the way the peptides are presented in the native protein.

Invitrogen offers the following options for peptide conjugation to carrier proteins: • KLH—keyhole limpet hemocyanin is a copper-containing protein, isolated from the hemolymphs of a mollusk. It exists in five different aggregated states (in Tris buffer pH 7.4), which readily dissociate with moderated pH change. Subunit molecular mass ca. 450 kDa. •BSA—bovine serum albumin, subunits of 67 kDa. Popular protein used in immunoassays to block non-specific binding sites. It should not be used as carrier if future assays involve BSA (for instance as blocking agent). •OVA—ovalbumin is a glycoprotein and the major protein of egg white. It has a pH of 4.6 and molecular weight of 45 kDa. •MAP—multiple antigenic peptide. This system uses peptides attached at many positions to a polylysine core. MAPs containing 2 to 16 copies of synthetic peptide molecule can be produced. MAPs have a high molar ratio of peptide antigen to core molecule and do not require the use of a carrier protein to elicit an antibody response.

MAP versus KLH • Eight copies of your peptide are synthesized on a branched lysine or MAP carrier core. • MAP peptides are suitable for direct injection (with adjuvant) for antibody production. • Since the peptide is linked through the C-terminus, the MAP technique favors N-terminal or internal peptides and is not recommended for peptides from the extreme C-terminus. The carrier attachment may also cause some steric hindrance. Use KLH conjugates for C-ter- minal peptides. • The average peptide length is about 15 residues. •MAP peptides behave much like large proteins. • MAP peptides may give a slightly higher titer due to their structure. Irrelevant antibodies can attach to the peptide branches.

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