Sulfhydryl-reactive keyhole limpet hemocyanin (KLH) for immunogen preparation with cysteine-containing peptides and haptens.

Thermo Scientific Imject Maleimide-Activated mcKLH products enable effective conjugation of sulfhydryl-containing peptide haptens to the highly immunogenic keyhole limpet hemocyanin (KLH) carrier protein for immunization and antibody production.

The high-quality mariculture KLH is rendered sulfhydryl-reactive by chemical modification with Sulfo-SMCC crosslinker, and the resulting maleimide-activated protein is then stabilized and lyophilized in phosphate buffer. Maleimide groups form covalent crosslinks with sulfhydryl (-SH) moieties on cysteine residues of peptides and other thiol-containing haptens. Numerous antigen molecules can be conjugated to each KLH molecule to make them highly immunogenic and increase the likelihood of eliciting immunized animals to produce high-titer antisera to the epitopes of interest. Select from among several package sizes of activated KLH protein, or choose the complete conjugation kit that includes optimized buffers and trouble-free spin desalting columns.

Highlights:

• Convenient kit – containing stabilized, lyophilized Maleimide-Activated mcKLH (in PBS with stabilizer) and accessory components to easily prepare ready-to-use immunogens for immunization
• Optimized activation – specifications require that each lot is activated with more than 400 moles of sulhydryl-reactive maleimide per mole of KLH, ensuring consistently high levels of conjugation
• Sulfhydryl-reactive – engineered for conjugating cysteine-containing peptide antigens to make effective immunogens for high-titer antibody production
• Simple, rapid procedure – complete the hapten-carrier conjugation reaction in two hours to yield sufficient immunogen for several injections and booster immunizations of host animals
• High-quality carrier protein – KLH is isolated from the mollusk Megathura crenulata (keyhole limpet); mcKLH is a high-quality form of KLH that is harvested from limpets grown in mariculture
• Highly immunogenic – KLH has a high molecular mass (4.5 x 10⁵ to 1.3 x 10⁷ Daltons; aggregates of 350 and 390 kDa subunits) and elicits a stronger immune response than BSA or ovalbumin
• Excellent solubility – our preparations of mariculture KLH are more soluble than typical forms and we also offer a PEGylated form for even greater hydrophilicity and solubility

Product Details:

Carrier proteins are large, complex molecules capable of stimulating an immune response upon injection. Successful production of antibodies specific to small antigens (i.e., peptides or drug compounds) requires that these haptens be covalently conjugated to a larger, more complex molecule (usually a protein) to make them immunogenic. Carrier proteins are chosen based on immunogenicity, solubility, and whether an adequate level of hapten-carrier conjugation can be achieved.

Keyhole limpet hemocyanin (KLH) is widely used as a carrier protein for conjugation to haptens and other antigens to make them more immunogenic for the purpose of antibody production. Because of its large mass and complexity, KLH elicits a stronger immune response than other carrier proteins. Because the protein is derived from a mollusk, it is phylogenetically distant from mammalian species and less likely to produce antibodies that will cross-react with typical target samples in assays.

Structure and conjugation reaction scheme of Thermo Scientific Imject Maleimide-Activated mcKLH. High-quality mariculture KLH is modified with Sulfo-SMCC crosslinker to produce the maleimide-activated carrier protein, which is supplied lyophilized. Addition of peptides or other molecules containing reduced sulfhydryl groups results in efficient and specific reaction to form stable thioether bonds. Each carrier protein molecule contains several hundred maleimide activations, allowing conjugation of many peptide hapten molecules per KLH molecule.
Structure of Thermo Scientific Imject PEGylated Maleimide-Activated mcKLH. Inclusion of polyethylene glycol (PEG) in the spacer arm of added maleimide groups increases the solubility and hydrophilicity of the carrier protein. Learn more... Polyethylene glycol and PEGylation Properties and performance of different forms of KLH and other carrier proteins

References:

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2. Hermanson, G.T. (2008). Bioconjugate Techniques. 2nd edition, Academic Press, New York. (Product #20036). Chapter 19 discusses carrier protein uses and the maleimide-activation chemistry.
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Related Products and Resources:

Protein Methods Library article – Antibody production and immunogen preparation
Example Product Data – Activation, conjugation and immune-response data for Imject Carrier Proteins

SulfoLink Immobilization Kit for Peptides – for preparing an affinity column with a cysteine peptide
Keyhole Limpet Hemocyanin (non-activated KLH) – for conjugation through amines or carboxylates
BSA and other Carrier Proteins