Peptides can be used as effectively as proteins to produce antibodies, polyclonal and monoclonal antibodies, usually with titers higher than 20000. Peptide antigens can't act as immunogens unless they bind to proteins. The production of high quality anti-peptide antibodies depends on the selection of peptide sequence, the success of peptide synthesis, peptide-carrier protein coupling, humoral immune response of host animals, adjuvants used, peptide dosage given, injection methods and purified antibodies.
Antibody is the most widely used protein recognition reagent in many biochemical fields because of its high specificity and binding ability. Natural or recombinant proteins are traditionally used to produce antibodies. The production of polyclonal antibodies against a protein produces antibodies against multiple epitopes, thereby maximizing the chances of recognizing the protein. However, this antibody pool does increase cross-reactions with other proteins. In contrast, the production of polyclonal antibodies against synthetic peptides produces antibodies against the target protein. In some cases, peptides are a better choice than proteins, such as raising antibodies to specific protein subtypes or phosphorylated proteins, and where proteins are not available.
We combine unique and powerful bioinformatics algorithms to design and analyze peptide antigens to ensure that each project produces customized highly specific antibodies with high titers. Our design service covers the purity level, amino acid composition, length, hydrophobicity and secondary structure of peptides. In addition, difficult amino acids are considered in the design to avoid them or for special treatment.
In general, we recommend using a peptide sequence of 8-20 amino acids to prepare antibodies. If the peptide is too short, its specificity is unlikely to be enough to enable the resulting antibody to recognize the target natural protein with sufficient affinity. On the contrary, sequences with a length of more than 20 amino acids may lose their specificity and induce secondary reactions.
For antibody production and testing, peptide purity > 70% is sufficient, but for biological activity studies, peptide purity > 95% is necessary. We can develop peptides with different levels of purity and has the ability to synthesize peptides with a purity of more than 98%.
The amino acid composition of the peptide strongly affects its solubility. Ideally, the content of hydrophobic amino acids should be kept below 50%, and there should be at least one charged residue (Arg, Asp, Glu or Lys) for every 5 amino acids.
To make antibodies against new epitopes, post-translational modification sites, and exon bridges, the possible sequence selection is limited, and when it is limited that insoluble sequences must be used, we will add spacers and hydrophilic residues.
Amino acid composition controls all aspects of peptide function. Peptide antigens should be designed to contain sequences found on the surface of natural proteins. Hydrophobic and hydrophilic residues should be included. In particular, it is beneficial for peptide sequences to be mixed with antigenic amino acids. On the other hand, our design process avoids problematic amino acids. In particular, cysteine, methionine and tryptophan are easily affected by oxidation and side reactions. Peptides containing multiple of these residues are also difficult to obtain with high purity. Sometimes, peptides are chemically modified or conjugated with specific ligands to obtain the desired properties.
Peptides of about 4 kDa are usually large enough to trigger an immune response, but smaller peptides need to bind to larger carrier molecules, such as keyhole hemocyanin, ovalbumin or thyroglobulin. The most common strategy is to add cysteine to one end of the peptide and use its mercaptan group to bind to the carrier molecule through maleimide crosslinker.
In the process of peptide synthesis, the formation of β-sheet will lead to the increasing incomplete solvation of peptides, resulting in a high incidence of missing sequences in the final product. We will choose areas with strong antigenicity, good hydrophilicity, good flexibility, high surface accessibility and turning angles, and avoid areas with α-helix and β-folding.
Peptides serve as a critical tool in antibody production by acting as antigenic sequences that trigger immune responses. They are often used to create both polyclonal and monoclonal antibodies, with specific advantages over traditional protein antigens in targeting precise epitopes or modified protein subtypes.
The peptide sequence is fundamental in generating highly specific antibodies. The selection of the right amino acid sequence, hydrophobicity, purity, and length are key factors in ensuring the resulting antibodies recognize the target with high affinity and specificity.
For optimal antibody production, peptide sequences of 8-20 amino acids are recommended. Shorter peptides may lack specificity, while longer peptides may reduce specificity and cause unintended immune responses. Our design process ensures the right balance to generate effective antibodies.
Peptide purity directly impacts the quality of the produced antibodies. For antibody generation, a purity level of 70% is acceptable, but for biological activity studies, peptides with >95% purity are preferred. Creative Peptides can provide peptides with a purity of >98%, ensuring high-quality results.
Peptides should contain a balanced mix of hydrophobic and hydrophilic amino acids, with at least one charged residue (e.g., Arg, Asp, Glu, or Lys) for every 5 amino acids. Our design process avoids problematic amino acids like cysteine, methionine, and tryptophan, which can cause oxidation and side reactions.
Solubility is crucial for peptide synthesis and antibody production. Peptides with high hydrophobicity or improper composition can be insoluble, affecting the quality of antibody generation. Creative Peptides uses spacers and hydrophilic residues to ensure solubility and improve the overall antibody production process.
Small peptides often need to be conjugated with larger carrier molecules, such as keyhole limpet hemocyanin (KLH) or ovalbumin, to stimulate a strong immune response. Our approach includes adding cysteine residues to the peptide for efficient conjugation to carrier proteins.
During peptide synthesis, we avoid the formation and other secondary structures that can cause aggregation and incomplete solvation, leading to missing sequences. Our design selects flexible, accessible areas of the peptide sequence, ensuring high-quality, functional peptides.
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