Frequently Asked Questions

This will depend upon the specific applications and requirements of your experiments.

Polyclonal antibodies will recognize multiple epitopes of an antigen. They are also more likely to maintain recognition of antigenic epitopes even when modest changes in conformation or aggregation occur. Polyclonal antibodies are also capable of recognizing different epitopes with different affinities. Because of this ability, polyclonal antibodies have a broader range of potential applications than monoclonal antibodies.

Monoclonal antibodies recognize only one epitope of the antigen and are highly specific to that particular antigen. They generally yield much lower background because of this specificity. Monoclonal antibodies also are highly homogenous and allow for unlimited production with sustained specificity.

Abgent's immunization protocols can be adapted for native proteins, recombinant proteins, and peptides. If the synthetic peptide is smaller than 8 kDa, we conjugate the peptide to KLH through the sulfo bond on amino acid cysteine. Antigens harmful to human health are not accepted.

Usually GST (Glutathione S-transferse) and MBP (maltos-binding protein) tags are immunogenic simply because they are large proteins that contain many epitopes. It is preferable to use antigen proteins without GST or MBP tags. However, other small tags, such as His-tag, Myc-tag and FLAG-tags, do not interfere with specific antibody production.

Our technical staff is highly experienced in antibody development and production, and we are pleased to consult with you on peptide design at no additional charge.

Please see our peptide design tips (hyperlink) for general design guidelines. Peptide sequence is crucial to creating an antibody that will specifically recognize native proteins with high affinity. We usually use peptides that are 8 to 15 amino acids in length.

If the antigen is a peptide, 10 mg will generally be sufficient. Less may be needed if your peptide is highly purified. For affinity purification of an antibody, we will need an additional 5 mg of peptide, preferably in dry form.

If the antigen is a protein, 2-5 mgs at a concentration of 0.5-1 mg/mL is generally sufficient. Be sure to let us know which buffer you included and the protein concentration. We recommend 3-5 mg at a concentration of 0.5 mg/ml or greater. For affinity purification of an antibody, 5 mg of soluble protein is required to prepare the immunoaffinity column.

This applies to both monoclonal and polyclonal antibody development.

Abgent offers a wide variety of custom services, including peptide synthesis and conjugation, as well as gene cloning and protein expression/purification using expression systems of either bacteria or baculovirus.

Abgent accepts either peptides or proteins for antibody development. Avoid additives that are toxic to animals, e.g. protease inhibitors such as PMSF, sodium azide etc. Peptides should be in dry form unless it is conjugated with a carrier protein (such as KLH or BSA). In such cases, the peptides should be in a buffered solution (no ionic detergents) with a concentration of at least 1 mg/mL. Proteins should be provided in buffer solutions (no ionic detergents), as a lyophilizate, or as a polyacrylamide gel slice. Destain gel in water to remove acids that can contribute to further denaturation of the protein.

Send samples overnight in a conical tube with an ice pack. Do not freeze samples, as this will interfere with the process.

Our custom monoclonal antibody development is performed in BALB/c mice, and polyclonal antibodies are produced in New Zealand rabbits.

Antibody titer is determined by ELISA. In this method, we coat 96-well plates with antigen and wash repeatedly to remove unbound antigen. When a KLH-conjugated peptide is the immunogen, we use unconjugated free peptides as a test antigen in ELISA. Serial dilutions of antibody are added to the antigen-bound wells and washed repeatedly to remove unbound antibodies. A labeled secondary antibody is then added and the number of bound primary antibodies is determined by colorimetric assay.

All of Abgent's antibodies have been extensively tested by ELISA and western blot. We can test antibodies in other assays, like immunoprecipitation, flow cytometry, and immunohistochemistry, upon customer request.

Purified antibodies are supplied in PBS with a low level of preservatives. The composition of PBS is 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4 at PH7.2. Anti-sera are supplied in original liquid form without preservatives. Antibodies can be stored at -80 ^o C for several years. Multiple freezing and thawing can denature protein, therefore, aliquoting for single use is highly recommended.

For details on project steps, project time periods and costs, please click on the following link: Antibody Development

You will need to provide us with a sequence of your peptide. We will evaluate it to see if it meets our requirements. See Peptide Order Form .

Our technical staff is highly experienced in antibody development and production, and we are pleased to consult with you on peptide design at no additional charge.

We employ Fmoc-solid phase peptide synthesis.

Abgent supplies all peptides as lyophilized powders. The lyophilized product should be stored at -20 ^o C to maintain maximum stability for the long term. Before use, let the container and its contents warm to room temperature in a desiccator to avoid absorption of water. Storage at 4 ^o C is acceptable for short-term convenient stock. Once you reconstitute the peptide, in solution slow degradation reactions may take place over the long term, such as oxidation of Cys, Met, and Trp, as well as deamidation of Gln. In addition, cysteines can form intra and intermolecular disulfide bonds.

Abgent offers peptides purified by reverse phase HPLC chromatography. Please specify the level of purity needed on the custom peptide synthesis order form.

Our technical staff is highly experienced in antibody development and production, and we are pleased to consult with you on peptide design at no additional charge. Please contact us at tech@abgent.com .