Antibody production guide
Three crucial components for successful antibody production
Antibodies can be produced against a wide range of different antigens. Most commonly used antigens are proteins and synthetic peptides.
If you do not have access to a target protein but knows its full length sequence, a synthetic peptide derived from the protein sequence can be used for antibody production. By carefully designing a suitable peptide, antibodies raised against the peptide can be used to detect the protein in a target assay.
Some general key considerations which will increase the probability of generating a succesful working peptide antibody:
Agrisera is happy to help with design of a suitable antigen. The design is based on your criteria together with estimated suitability for synthesis, conjugation and immunization. If you are choosing a peptide yourself you are welcome to send us the sequence for a statement regarding immunogenicity and solubility. Read more »
Proteins (native or denatured) isolated from a tissue or recombinantly produced, are frequently used for antibody production. How the proteins are produced vary from case to case depending on access of suitable tissue and final use of the antibody. Basically, antibodies produced against native proteins tend to react best with native proteins (Immunoprecipitation) and antibodies produced against denatured proteins to proteins subjected to denaturing conditions (Western Blot, IHC). However, well functioning antibodies can be generated without necessarily follow these rules. Given that adjuvants can have a degenerative effect on the protein, the guidelines are also not entirely obvious. The purity of a protein is also a factor to consider. The higher purity, the greater the proportion of specific antibodies will be. If the protein is impure, it can be separated on SDS gel and the gel piece can be used for immunization. If the protein is expressed with a fusion partner, antibodies will be generated against the fusion partner as well and it might be necessary to find a strategy to remove those non-specific antibodies. This can be done by cleavage of the tag prior to immunization or by affinity purification.
Are you are interested in producing antibodies against a protein? Agrisera offers a full service for production of high quality recombinant proteins. Read more »
What will give better results, immunization with a peptide or a protein? Each of these approaches has advantages and disadvantages. Antibodies bind to specific epitopes on a target protein and the epitopes are two to three amino acids in a row (linear epitope) or distant amino acids brought together in a 3D structure. Antibodies against proteins or protein fragments will recognize several epitopes compared to peptide antibodies that are considered to be mono-specific as they target a more limited epitope. Anti-peptide antibodies are thus preferred if cross-reaction with other protein family members needs to be avoided. Anti-peptide antibodies can on the other hand increase the risk for possible cross-reaction, if the chosen sequence has high homology with other arbitrary proteins.
Approaches to consider:
Finally, preparation of fine emulsion of antigen and adjuvant is another important factor to secure efficient antibody production. With 30 years of experience, Agrisera has developed a good method to achieve this.
Keeping animals in natural conditions will make them healthy and allow stimulation of their immune response. Each animal produces, prior to immunization, millions of antibody combinations that can fit potential proteins to be encountered in the future. If clones producing antibodies to the desired antigen are not present already or present in very low amounts it might be difficult to obtain a good response. Thus, it can be beneficial to use more than one animal per antigen. When a number of animals are immunized with the same antigen, resulting antibodies will bind to similar, but not identical epitopes. Therefore antibodies produced in two animals can in some cases be used with different success in various techniques, e.g. one works better in a western blot, another in immunoprecipitation.
Choice of species?
It is important to evaluate the antibody in your own assay to see how it works. During the time the antibody is produced, all necessary preparations can be done. Proteins can be blotted in advance and stored dry in RT for up to six months. When you evaluate the antibody we recommend using:
Re-using of a primary antibody solution during initial testing is not recommended, especially not if you work with quantitative Western Blot.
If suitable controls are difficult to obtain, saturation of antibody with peptide/protein used for immunization can be an alternative. In case of proteins with low tissue abundance, it is of crucial importance to use sensitive detection systems based on chemiluminescence, such as Agrisera ECL Bright or to work with a specific tissue fragment or fraction.
If you need any help with immunological techniques, you are welcome to visit our western blot trouble-shooting or contact us with specific questions.