intavis AG | FAQ - Frequently Asked Questions

If you want to have an antibody that is directed against a single peptide out of a protein sequence, the "Custom Antibody Service" is the right package to choose.

The "Multi-Epitope Service" yields a polyclonal serum raised against two or more epitopes simultaneously. This procedure is more economical than several different immunisations with individual peptides from the same protein.

Why anti-peptide antibodies?

We think that anti-peptide antibodies against carefully chosen peptides are more specific than antibodies generated from longer sequences (e.g. recombinant proteins or parts of whole proteins). For recombinant sequence parts of proteins you will never know if polyclonal antibodies are generated against epitopes that are located inside the natural folded protein. For peptides there are some algorithms that help to choose sequences that have a high potential to be located at the surface of the protein.

How to choose the right peptides for immunisation?

We can assist you in choosing the right peptides by analysing your protein sequence and recommend the most immunogenic peptide sequences. This service will not be charged.

In general, antigenic epitopes are hydrophilic, flexible and located at the surface of proteins. The C- and N-terminus of proteins are often very flexible regions and therefore good targets for antibody production. However, if these sequences are too hydrophobic (e.g. for membrane proteins) it is advisable to choose a different peptide sequence as antigen.

How long should the peptides be?

The peptide length should be between 8 and 18 amino acids. If the sequences are longer than 20 amino acids, the resulting antibodies may be less specific.

Why do peptides have to be conjugated prior to immunisation?

The size of most peptides is too small to generate an immune response. By conjugation of the peptide to carrier proteins (KLH, BSA or OVA) the size and the immune response increases.

What coupling chemistry can be used?

If the peptide has no internal cysteine, cysteine can be used at the N- or C-terminal end of the peptide to couple it via the sulfhydryl group to the carrier protein. The coupling is done with a heterobifunctional crosslinker.

What carrier protein do you usually use for conjugation?

In general KLH is the preferred carrier protein because BSA is often used for the blocking of surfaces (e.g. ELISA or microarrays). If the antibodies are not purified by a peptide column, both antibodies against the peptide and the carrier protein are in the serum. The latter could cause false positive signals if carrier protein and blocking agent are the same.

How do I store my antiserum?

The antibodies can be stored 4°C for a few weeks. For long term storage the serum should be stored at -20°C or -80°C. Most antibodies stored in this way will remain stable for several years. To avoid repeated freeze-thaw cycles it is recommended to divide the serum into aliquots.

Sodium azide as antimicrobial agent can be added to the serum to avoid bacterial contamination of the serum.

Should I add sodium azide to the antiserum?

We recommend adding sodium azide unless the serum is directly used in cell culture assays. Sodium azide prevents bacteria from growing as they can produce proteases that denature the antibodies in the serum. It can be removed by dialysis with a membrane that has a molecular weight cut-off of 13.000.

Note: Sodium azide is toxic to most organisms and should be removed for some applications.