Carrier conjugated peptides

Immunization with naked peptide antigens is very often not successful, as peptides are often too small to generate significant immune responses by themselves. In order to solve this problem, peptides should be conjugated to large carrier proteins, such as Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA) or Ovalbumin (OVA). To ensure the effective and successful generation of anti-peptide antibodies, the use of peptide – protein conjugates is always recommended.

Peptide – Carrier protein coupling step by step

1. Selecting the carrier protein

Researchers may select a carrier protein form a range of options, of which the most commonly used carrier proteins are the following:

  • KLH peptide – Keyhole Limpet Hemocyanin (MW 4.5×10⁵ – 1.3×10⁷ Da) is the most commonly used carrier protein, most likely because it has a significantly higher immunogenicity as compared to other proteins. KLH-conjugated peptides often have limited solubility in water, which results in a cloudy appearance. The turbidity does not affect its immunogenicity and the resulting solution can be used for successful immunizations.
  • BSA peptide – bovine serum albumin (MW 67×10³ Da) is one of the most stable and soluble albumins available. It contains 59 lysines, of which approx 30-35 are accessible for conjugation. Therefore BSA is a popular carrier protein for weakly antigenic compounds.
  • OVA peptide – ovalbumin (MW 4.5×10⁴) is a protein isolated from chicken eggs. It is often used as a control carrier protein to verify that antibodies are specific for the target peptide rather than the carrier protein itself (e.g. KLH/BSA).

Conjugation to other carrier proteins (e.g. HSA, Thyroglobulin) is available upon request.

2. Peptide design & synthesis

Depending on the linkage chemistry to be used, we synthesize your peptides with the appropriate chemical handles at the right position, such that the peptide gets optimally exposed at the surface of your protein carrier. If desired, we can also introduce a rigid or flexible spacer to optimally finetune the distance to the carrier surface.

3. Linkage Chemistry

In most cases we apply one of two different linkage chemistries for peptide-to-protein conjugation, i.e. the standard ‘maleimide-based’ technology or the more advanced ‘Hydralink’ technology.

The ‘classical-type’ linker connects to the protein via formation of a standard amide-bond to any free amine group (free lysine side chain or N-terminus), whereas the peptide gets connected to the linker via conjugate addition of a free thiol to a maleimide functionality. This provides chemically stable peptide-protein linkages that cannot be destroyed under standard laboratory conditions. There are various linkers available in this format that mainly differ in size (MCS, GMBS), rigidity (MCS vs. MBS), and water-solubility (MBS vs. sMBS).

Name Chemical structure Reactivity Å H₂O Solubility
N-(ε-Maleimidocaproyloxy) succinimide ester
amine + sulphydryl ~9.0 NO
sEMCS (= sulfoEMCS)
N-(ε-Maleimidocaproyloxy)sulfo succinimide ester
amine + sulphydryl ~9.0 YES
m-Maleimidobenzoyl-N-hydroxysuccinimide ester
amine + sulphydryl ~7.5 NO
sMBS (= sulfoMBS)
(m-Maleimidobenzoyl-N-hydroxy)sulfo succinimide ester
amine + sulphydryl ~7.5 YES
Maleimidobutyryloxy succinimide ester
amine + sulphydryl ~6.5 NO
sGMBS (= sulfoGMBS)
(Maleimidobutyryloxy)sulfo succinimide ester
amine + sulphydryl ~6.5 YES

Next to this, we also apply the ‘Hydralink’ technology that couples peptides with free aminoxy (RO-NH2) or hydrazino-nicotinamide (HyNic) groups to any desirable carrier protein. To this end, the carrier protein is first derivatized with the reagent ‘S-4FB’ or ‘sulfo-S-4FB’ that converts a free amino group at the protein surface into an benzaldehyde group, which reacts with the reactive aminoxy or HyNic groups on the peptide. The technology has an important advantage over the standard thiol-maleimide chemistry, because it can be quantified by UV-spectroscopy at 354 nm. Moreover, this chemistry leaves disulfide bonds untouched and completely intact and avoids the problem of SS-bond scrambling when using free thiols.

Name Chemical structure Reactivity Å H₂O Solubility
p-Formylbenzoyl-N-hydroxysuccinimide ester
amine + aminoxy ~7.5 NO
(p-Formylbenzoyl-N-hydroxy) sulfo succinimide ester
amine + aminoxy ~7.5 YES

4. Quality control

For conjugates where the HydraLink technology is applied, the peptide-protein ratio can be determined using UV spectroscopy.

The number of “free” 4FB units on the protein can be quantified in a reaction with 2-Hydrazinopyridine, a compound that absorbs at 350 nm. Determining the number of free 4FB before and after peptide conjugation yields the average peptide/carrier ratio.

For final protein concentration, a BCA assay is performed. This is a high-precision protein assay for determining protein concentration. The assay provides accurate calculations with most sample types encountered in protein research.

These additional analyses are available upon request.

Note that these analyses are less reliable for conjugates, especially those related to KLH, turned turbid after a protein-peptide reaction.


5. Delivery

The conjugates are supplied in phosphate buffer as frozen solutions. They are transported on dry ice.

For short-term storage, -20°C is preferred.
For long-term storage, -80°C is preferred.

We use cookies to ensure that we give you the best experience on our website. If you would like to, you can change your cookie settings in your browser.
For more information check our privacy policy
Find out more
Accept All Cookies
By continuing to browse or by clicking "Accept All Cookies" you agree to the storing of first and third-party cookies on your device to enhance site navigation, analyze site usage, and assist in our marketing efforts.
Cookie Policy
Cookie Settings
Accept All Cookies