Antigen Preparation
- How much protein do I need to send?
- We recommend sending 2-3 mg of the antigen for a rabbit or chicken project and 5-7 mg for a goat project. We recommend .5 mg/ml as a minimum protein concentration and welcome higher concentrations.
- How should I send my protein to Pacific Immunology?
- Please ship the antigen in a Styrofoam box with ice packs (dry ice isn’t necessary) via FedEx, UPS or DHL Overnight to the address listed on our Order Form. To avoid additional shipping charges by these companies, please insert the Styrofoam box into a cardboard box or shipping pak prior to shipping. Please also include a printed copy of the Order Form with the shipment.
- Does the protein need to be soluble?
- No, insoluble proteins can be used for immunizations.
- Can I generate antisera against gel strips containing my protein?
- Yes, immunizing with gel bands works well for generating polyclonal antisera against a purified protein. If providing a gel strip, please stain with coomassie blue, de-stain, rinse and cut out the strip containing the target protein. Please avoid freezing or homogenizing the gel strip so that we can aliquot appropriate quantities of the protein for each immunization. With gel bands in particular, it is important to maintain a higher concentration of the protein as outlined above.
- Should I generate antibodies against a full-length protein or a peptide?
- Generating antibodies against a full-length protein will provide a pool of antibodies against multiple epitopes from the protein, thereby maximizing the probability of recognizing the endogenous protein in the target assay. However, this larger pool of antibodies does increase the possibility that some of those antibodies will cross-react with other proteins in the assay.
Immunizing with a peptide sequence, by contrast, allows the serum to be affinity purified against the peptide sequence, thereby permitting isolation of antibodies that are highly specific to the target protein. The only potential downside to this approach is that there is a risk that the chosen sequence won’t correspond to an exposed region of the native protein.
- Does Pacific Immunology offer antigen design assistance?
- Yes, our peptide chemists will analyze your protein sequence and recommend the most immunogenic sequences based on such factors as the hydrophobicity / hydrophilicity and folding characteristics of the protein. We have consistently obtained very good results with these recommendations and provide this service at no additional charge.
- Why is conjugation of the peptide to a carrier protein necessary?
- The molecular weight of most peptides is too small to generate an immune response in the animal. Conjugation to a carrier protein such as KLH will not only increase the size of the antigen, but increase its immunogenicity as well.
- What role does the adjuvant play and what is the difference between CFA and IFA?
- An adjuvant is a substance that, when combined with the antigen, serves to enhance the immune response against the antigen. Freund's adjuvants are the preferred adjuvant of choice for use in antibody production, and Pacific Immunology uses its own premium line of AdjuLite® adjuvants for all immunizations. AdjuLite® Complete Freund's Adjuvant is used only for the first immunization and contains mycobacteria, which stimulates the host's immune system. AdjuLite® Incomplete Freund's Adjuvant is used for all subsequent immunizations.
