Polyclonal Antibody FAQs
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, and rats 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.
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.
How do you choose the best antigen?
First, we discuss with you what your most important needs are (such as making sure the antibody does not cross-react with protein family members, etc.). We then use that framework to select a recommendation that is antigenic and available within the protein's 3D structure.
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.
What chemistry do you typically use for carrier protein conjugation?
We recommend adding a cysteine to one of the termini of your peptide (or designing the peptide to use one that is natural) and using the free sulfhydryl group to conjugate to KLH. This is a straightforward, highly efficient and stable chemistry that will not affect the specificity of the resultant antibody.
Several alternative conjugation methods are also available, but only recommended when you have internal or multiple cysteines in your antigen.
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. The measurement of the resultant signal, called a titer, is 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 Vivitide Standard Antibody Package provides ~80mL antisera. The Vivitide Affinity Purified Antibody Package often yields >10mgs of purified antibody 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 about 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?
We design the peptide so that it gives our antibodies a very good chance to be used across multiple applications such as western blots, staining, immunoprecipitation, flow cytometry, and ELISAs. Whether it is successful doing so depends on multiple factors, including not only the quality of the antibody that is made but also the specific systems and methods in place that are used for testing. Unfortunately, as these testing systems are entirely out of our control, we cannot make any guarantees that the antibody will work perfectly in the application that you need.
What are your accreditations?
We only contract with programs that are AAALAC-accredited, OLAW-assured, and USDA-licensed. Use OLAW # A3975-01 if you need to file your project through the NIH.What is Vivitide's policy of ownership and confidentiality?
The customer owns all antibody material produced under contract to Vivitide. Each order is handled with strict and complete confidentiality. We can also work together to execute a Confidential Disclosure Agreement (CDA).