Polyclonal Antibody

What type of antigens can be used for polyclonal antibody production?
Most molecules can be injected into animals to generate antibodies, but some molecules are deliver better results for specific applications than others. Full-length proteins, either expressed recombinantly or purified from natural sources, are the best immunogens for recognizing full length proteins (ex: IP, IHC). Peptides can be easier and cheaper to produce and are well suited for assays such as ELISA and Western Blotting.  There is a risk that anti-peptide antibodies might fail to recognize the full-length protein from which they were derived. Small molecules tend not to be immunogenic due to their small size and must be conjugated to carrier proteins before they can be recognized by the immune system.

Why should I use a peptide for antibody production?
Anti-peptide antibody production is a useful tool as it allows one to focus on a specific region of a protein. Your resulting antibody may end up more specific, as you can completely regulate exactly what region of the protein the antibody is made against. Using a peptide also increases the chance of successful antibody production, especially on proteins that have an overall hydrophobic trend. Because we can hand select the region that the antibody will be produced against, we are given the freedom to select regions that are particularly antigenic and specific, increasing your chances of a successful project.

Which antibody package is right for me?
Polyclonal antibodies can be unpredictable in their specificity and concentration. If you are doing a quick non-sensitive assay, it may be possible to use the antisera directly without any additional purification. In this case, order our Standard Antibody Package. If you require a more specific and concentrated antibody (such as for extensive western blotting or staining applications), the Affinity Purified Antibody Package is a more appropriate choice. A good example where affinity purification is helpful is the production of a phospho-specific antibody.

What species should I choose for my antibody production?
Rabbits are the industry standard as they are highly reliable, affordable, and respond in a reasonable amount of time. Should you need a large volume of serum, goats or sheep are an excellent option. Chickens are ideal for proteins with high homology in mammals. Guinea pigs, hamsters, rats and mice are great options if you need an antibody to do co-localization work with a rabbit antibody.

Why do I need two rabbits?
Every animal responds differently to immunization and often animals make different subsets of antibodies to a single antigen. Immunizing two animals increases the odds of one of the animals providing antibodies with high reactivity and specificity to your protein of interest.

How do you choose the best antigen?
Our custom antibody development scientists take care in designing each of the projects that we produce to maximize success.  We make sure your peptide is antigenic, available within the protein's three-dimensional structure and has the ends appropriately blocked (with acetylation and amidation) to mimic the natural structure of the protein in vivo. We also recommend adding a cysteine residue to the N- or C-terminus in order to link the peptide to a carrier protein or the affinity matrix without compromising its structure. Any other details that need to be addressed (specificity between species, protein family members, etc.) are also taken into account.

Can I inject more than one peptide into the same set of animals?
Injecting multiple peptides into one set of animals is a useful, cost efficient option that increases your overall chances of success if you only need an antibody to one of the two sequences injected (if they are to the same protein, for example). Often one peptide will elicit a much stronger response than the other, so we cannot guarantee that both peptides will work.

Why does my KLH/peptide solution appear cloudy?
KLH (Keyhole Limpet Hemocyanin) is a large (MW = 4x105 - 1x107 kDa) aggregating protein. Because of its size and structure, its solubility in water is often limited. This can manifest as tendrils or a general cloudy appearance. This does not affect antigenicity, and the turbid solution can be used for immunizations.

What do I do with my pre-immune serum?
Pre-immune serum is a great negative control for a wide variety of experiments.

How do you test for reactivity?
The serum from the first production bleed is tested for reactivity against the peptide using an enzyme-linked immuno-sorbent assay (ELISA). The peptide is coated onto a 96-well plate, followed by serial dilutions of antiserum that contain the antibody. This titer measures the amount of specific antibody in the sample. The results are then sent to you with your shipment.

How much antibody should I expect to receive?
Antibody production depends on epitope selection, peptide synthesis, carrier conjugation, immunization procedures, and biological systems in animals. Therefore, there is wide variability in the antibody response. The Abpro Standard Polyclonal Antibody Package provides ~80mL of antisera. The Abpro Affinity Purified Polyclonal Antibody Package often yields 5-10mgs of purified antibody (or more) in addition to 40mL antisera.

How do I store my antisera / antibody?
Sera storage should be at -20° C (for up to one year), or 4° C for less than a month. For longer term storage, add 0.05% azide and freeze. Purified antibody is most stable when stored at -80° C and multiple freeze-thaws are avoided. Alternatively, you can add glycerol to a total concentration of 30-50% and store the antibody at -20° C.

How long can I expect my rabbits to produce good antibody?
With the inherent variability in polyclonal antibody systems, this is always difficult to predict. As long as the initial response is good, your rabbits should keep producing roughly the same grade antibody for an average of six months. Many rabbits are productive for longer than one year, but the strength and specificity of your signal can fluctuate during this time. The longer the rabbits are alive the better they acclimate to the antigen, and the response begins to decrease.

Will I be able to use my antibody in multiple applications?
This depends on multiple factors, most notably the systems and methods in place for testing and the quality of the antibody that is made. We are proud to say that we make excellent antibodies, and that we actually design them to be used in applications such as western blots, staining and ELISAs. Due to the variation in testing systems, however, we cannot make any guarantees that the antibody will work perfectly in the application that you need.

What are your accreditations?
Our polyclonal antibody facility is AAALAC accredited, OLAW assured, and USDA licensed to ensure all proper care is taken and research animals are treated appropriately.