Frequently Asked Questions
If sending in an antigen for immunizations, projects typically start within one week of receipt. If you order a peptide antigen synthesis from ProSci, synthesis begins within 1-2 business days of order receipt. Purifications start the Monday after receipt as long as samples are received by the previous Thursday.
Please ship the antigen attention Antigen Receiving to ProSci Incorporated, 12170 Flint Place, Poway, CA 92064, USA.
Expect to receive an email confirmation upon receipt of the antigen along with an expected timeline for the project to start. Once immunizations begin, a project schedule is emailed along with expected ship dates of the materials.
This is a great reason for taking advantage of our free antigen design assistance. If you choose one of the sequences we recommend for peptide synthesis, we will guarantee an immune response to the antigen by ELISA titration in at least one of the rabbits in our custom rabbit polyclonal antibody packages.
For polyclonal antibody services, invoices are generated the week that animals are immunized, after peptide synthesis.
For monoclonal antibody services and recombinant antibody services, invoices are generated at the start of each phase.
For custom polyclonal antibody development and production, we offer mouse, rabbit, chicken, goat, and llama.
For custom monoclonal antibody development, we offer mouse, rabbit, and llama.
For custom monoclonal antibody production, we offer in vitro production with murine hybridomas as well as recombinant antibody production using bacterial or mammalian expression systems for llama single domain antibodies and rabbit monoclonal antibodies, respectively.
A standard two rabbit schedule uses 1 mg of antigen (peptide, protein, etc.), but 1.5-2 mgs of protein are recommended, especially if you require ELISAs or extensions. For affinity purification, 2-3 mg of lyophilized peptide and 3-5 mg of soluble protein are required. We prefer a concentration of 1 mg/ml or greater. Check out our Antigen Requirements page to learn more.
This is not a problem; send us what you have ready and we will start your project. You can send us more as it becomes available.
Approximately 10-12 kDa is necessary for an immune response. Anything smaller needs to be coupled with a carrier protein, typically KLH, so that the immune system can recognize it.
- DNA plasmid: ProSci can use a DNA plasmid to generate antibodies; however, you need to provide an immunization protocol (bleed/boost schedule and quantity of plasmid to be immunized, etc.) because different plasmids require different schedules. Immunizing with a DNA plasmid is different from immunizing with a protein or peptide antigen. The plasmid can be shipped to us on blue ice.
Enzymes: Enzymes can easily be used for antibody production; we need to know concentration in order to administer immunizations and to perform ELISAs.
- Cells: Cells (i.e.: isolated from an organ like the spleen) can be used as an antigen, but we cannot perform an ELISA to determine titer. However, we can screen and select for monoclonal or single domain antibodies using flow cytometry. Blood cells must be washed away before we can accept the cells as an immunogen.
- Cell Lines/Lysates: Cell lines and lysates can be used as a polyclonal antigen; however, they cannot be used for a monoclonal antigen as screening via ELISA with cells is not available from ProSci at this time. A lysate would work as an immunogen, but it would contain so many different proteins that the antibody wouldn't be very specific. However, these may be ideal for monoclonal and recombinant antibody development, depending upon project goals.
Note: We cannot use any living organisms for immunization. If the recombinant organisms are dead, and contain no toxic materials harmful to the animals or personnel, they could be used as an immunogen. If you cannot find the type of antigen you would use in this list, please contact our custom antibody experts at email@example.com for more information.
Yes, and its FREE. Our scientists will help you select a sequence that has the potential to raise a good antibody response. Just email your full length protein sequence(s) along with any supplemental information such as accession number and desired final application to firstname.lastname@example.org and we will have a response in 1 to 3 business days.
In the most general terms, peptide antigens need to be around 70% purity or greater and protein antigens need to be at least 90% purity or greater. The high purity requirement comes from 2 main points:
Protein antigens should be of a higher purity because the manufacturing process can yield other unrelated proteins which will cause different immune responses. With synthetic peptide antigens, the impurities tend to be fragments of partially synthesized peptides vs. something completely unrelated to the desired target. Partially synthesized peptide antigens may still be sufficient for generating the desired immune response.
The purer the antigen, the more precise the host’s immune response will be. Any impurities in your antigen run the risk of being immunoreactive as well, generating undesirable antibodies that could cause high background.
The necessary antigen purity also depends upon what you are trying to do with your project. For polyclonal antibodies, >70% peptide antigen purity is fine, but for monoclonal antibodies, >90% antigen purity is the standard.
Additionally, peptide antigens containing modified amino acids should also be >90% purity even for polyclonal antibody development.
Though these are reasons to try and provide very pure antigens for the best results, this does not mean we will not accept lower purity antigen. A custom project is just that: custom. We will still generate an antibody for you if you provide us with 60% pure protein, but you may not obtain the desired antibodies at the end. With antibody development and production, what you put in is what you get out.
Please cleave the larger tags (GST, MBP, etc.) as they can interfere in specific antibody production. Small tags such as His, Myc, and FLAG do not need to be cleaved prior to antibody production.
Sure, however gel strips can only be accepted for our Serum Package. Gel strips cannot be used in conjunction with ELISA and affinity purification as soluble protein is needed. Additionally, the protein should be reasonably concentrated prior to running on the gel (2-5 mg/ml). It is recommended to run 100-200 µg per lane to minimize the amount of gel. Just stain the whole strip with coomassie blue, then de-stain, rinse, and cut the desired strip from the gel (please don't "chop it up"). After you cut the band from the gel, place it in a 15 or 50 ml conical tube with an ice pack- don't freeze it.
Use 5% methanol, 7.5% acetic acid, and fill the remainder with ddH2O. De-stain gel in 100 ml solution overnight and replace with fresh de-stain if needed. The gel will remain blue even though the coomassie dissociates from the proteins.
How are you currently storing your antigen? You only need dry ice if the storage conditions are -80°C. If the antigen is stable, ice packs are fine. Please ship it overnight via FedEx or a similar carrier.
- Purified Antibodies: We recommend storing purified antibodies at -20°C for long-term storage, but aliquot into smaller volumes to avoid multiple freeze-thaw cycles. Stored frozen, antibodies can be stable for several years. For short-term storage of several months, purified antibodies can be stored at 4°C. The antibodies are provided in PBS with 0.02% sodium azide.
- Peptides: Store unconjugated peptides at ‐20°C; stable for several years when lyophilized. Peptides in solution should be aliquoted to avoid multiple freeze‐thaw cycles.
- Serum: Aliquot into single‐use volumes so that the entire quantity is not exposed to multiple freeze‐thaw cycles. Store serum at ‐20°C; stable for several years. Samples that are in use may be stored at 4°C for up to two weeks. The provided serum does not contain sodium azide. 0.1% can be added as a preservative to prolong the serum shelf‐life, but sodium azide may interfere with some assays.
- Yolks: Isolated yolks are stable for several months at 4°C. Adding 0.1% sodium azide will help preserve the yolks. It is best to isolate the IgY from the yolks for long‐term storage instead of storing the yolks as‐is. Yolks can also be stored at ‐20°C, but lipid removal and purification becomes more difficult after freezing.
- Affinity Purification Column: Store upright (blue or green cap up) in a refrigerator at 4°C. Columns can be reused multiple times without significant loss of activity. Columns with “SL” on the label are stable for years, but ones with “S4B” may be stable for only one year.
- Hybridoma Cell Lines: These should be stored in liquid nitrogen. For short term storage of a few days, cells can be stored at ‐80°C, but this may reduce the viability and chance of recovery.
- Supernatants: Hybridoma supernatants may be stored at 4°C short-term, but should be stored at -20°C for long-term storage. Recombinant antibody supernatants (rabbit monoclonal and llama single domain) should be stored frozen at -20°C.
- DNA/Plasmids/Glycerol Stocks: Final recombinant antibody clones should be stored frozen at -20°C.
The peptide/protein is bound covalently to CNBr activated Sepharose 4B or SL gel in a column. The antibodies within the polyclonal pool that are specific to this antigen are allowed to bind. The unbound antibodies and other serum proteins pass through the column, and the antigen-bound antibodies are eluted. The resulting purified antibody is highly specific. ProSci will provide you with the serum, flow through, purified antibody (a yield from 0.1 mg to 0.5 mg per ml of serum), reusable column to the antigen of interest and ELISA results.
For all polyclonal antibody projects, the OD values for the sample tested are reported and summarized as a titer (defined as the dilution where the OD value is equal to 0.100). Titers >5,000 are typically indicative of an immune response against the sample.
As long as we have a sample to coat on an ELISA plate, we will run an ELISA on the antiserum, purified antibody, and immunodepleted serum (i.e. flow through). This data provides several pieces of information, such as the purification efficiency and whether there is additional antibody in the flow through. For purification efficiency, look at the OD values for each dilution between the serum and purified antibody and look for where the signal gets close to 0.100 in each and if this is at 1:25,000 in the antiserum and 1:125,000 in the purified antibody, then the antibody has been concentrated 5-fold.
Ideal ELISA results for a phospho-specific project are to have a high titer against the phosphorylated peptide and a low titer against the non-phosphorylated peptide. However, an ELISA on the antiserum does not correlate to purification success in being able to isolate antibodies specific to the phosphorylation or how the purified antibody would characterize in other assays.
For the affinity purification, we also run an ELISA against each antibody and each peptide. As such, there are four sets of data – non-phospho peptide with the non-phospho antibody, non-phospho peptide with the phospho antibody, phospho peptide with the non-phospho antibody, and phospho peptide with the phospho antibody. From all this data, focus on the phospho antibody against each peptide. Again, look for a higher signal against the phospho peptide than the non-phospho peptide.
While an ELISA is a great measure to determine whether there is an immune response against the antigen, it unfortunately cannot predict how the antisera or antibody will work in other assays. Even though there may be a high immune response, an antibody may not work for other applications such as Western blot, flow cytometry, immunocytochemistry, immunoflourescences, or immunohistochemistry.