The basic principles of antigen polypeptide selection are (1) as far as possible on the protein surface; (2) Ensure that the sequence does not form α-helix; (3) The peptides at the N and C terminals were better than those in the middle; (4) To avoid the sequence within the protein repeating or close to the repeating segment; (5) Avoid peptides with strong homology; (6) Cross-linking can be done at the N and C ends, and the selection is based on the fact that the cross-linking is at the less important end for antibody production; (7) The sequence should not have too much Pro, but one or two Pro is good, can make the peptide chain structure relatively stable, beneficial for the production of specific antibodies.

For optimal antibody production, it is necessary to carefully design the antigen polypeptide to meet the basic condition that the antigen does not produce an overactive immune response during the immune process, but at the same time produces antibodies that bind to the protein of interest.

It is usually recommended that the sequence length of an antigenic polypeptide be between 8 and 20 amino acid residues. If the sequence length is too short, there is a risk that the polypeptide is too special and the affinity (binding ability) between the resulting antibody and the natural protein is not strong enough. Similarly, if the sequence length is longer than 20, it is possible to introduce secondary structure and the resulting antibody loses its specificity. Usually the synthesis difficulty increases, it is not easy to obtain high purity products.

To avoid the risk of the recognition region being hidden within the protein, it is usually recommended to select the N and C ends of the protein to produce the corresponding antibody. Because in a complete protein, the N and C ends are usually exposed to the surface of the protein. However, it must be noted that the C-terminus of the membrane protein is too hydrophobic to be suitable as an antigen.

The molecular weight (MW) of the peptide antigen (unbound) is too small to be used in traditional western blotting (WB) tests. To verify the specificity of target bands in WB, it is recommended to run an "antibody block" control and use peptide antigen to block the primary antibody. The recommended bleeding dilution is 1:1000 (equal to 1µg/ml) and the blocking peptide is about 2µg/ml.

A quality antibody is one that has the appropriate sensitivity and specificity to answer the questions your test poses. In addition, one antibody is not necessarily suitable for all applications, as each application has different performance specifications.

Both polyclonal and monoclonal antibodies have their own advantages. Generally speaking, monoclonal antibodies have strong specificity, but relatively small affinity, and relatively low sensitivity to detect antigens. The specificity of polyclonal antibody is weak, but the affinity of the antibody is strong and the sensitivity is high. Compared to monoclonal antibodies, large quantities of polyclonal antibodies are relatively quick and inexpensive to produce, and they are nonspecific because they are able to recognize multiple epitopes on any one antigen.

For qualified protein antigens, Qiangyao can guarantee two ELISA and WB positive clones against the immunogen. For qualified peptide antigens, Qiangyao can guarantee two positive clones, the ELISA results against the immunogen are positive. For other cases, Qiangyao will provide assurance based on the immune response after immunization.