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 Imject Maleimide-Activated Mariculture KLH and Kit 

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

Maleimide-activated KLH carrier protein for immunogen preparation with sulfhydryl peptidesThermo 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.
Maleimide-Activated KLH is prepared using Sulfo-SMCC, making it reactive to peptides with terminal cysteines.
   

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

Structure of PEGylated Maleimide-Activated mcKLH.

References:

  1. Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. (Product #15050). Chapter 5 discusses the use of carrier proteins.
  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.
  3. Lagace, T.A., et al. (2006). Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and inlivers of parabiotic mice. J. Clin. Invest. 116:2995-3005.
  4. Casais, R., et al. (2005). Gene 5 of the avian coronavirus infectious bronchitis virus is not essential for replication. J. Virol. 79:8065-8078.
  5. Becker, E.B.E., et al. (2004). Characterization of the c-Jun N-terminal kinase-BimEL signaling pathway in neuronal apoptosis. J. Neurosci. 24:8762-8770.
  6. Chang, S.C., et al. (1994). Cartilage-derived morphogenetic proteins. J. Biol. Chem. 45:28227-28234.
  7. Heymach, Jr., J.V., et al. (1995). The biosynthesis of neurotrophin heterodimers by transfected mammalian cells. J. Biol. Chem. 270:12297-12304.
  8. Joshi, H.C., et al. (1992). g-Tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature 356:80-83.
  9. Juge-Aubry, C.E., et al. (1995). Peroxisome proliferator-activated receptor mediates cross-talk with thyroid hormone receptor by competition for retinoid X receptor. J. Biol. Chem. 270:18117-18122.
  10. Karim, A.S. (1995). Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester. Nucl. Acid. Res. 23:2037-2040.
  11. Slunt, H.H., et al. (1994). Expression of a ubiquitous, cross-reactive homologue of the mouse ß-amyloid precursor protein (APP). J. Biol. Chem. 269:2637-2644.
  12. Wojcikiewicz, R.J.H. (1995). Type I, II, and III Inositol 1, 4, 5-triphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J. Biol. Chem. 270:11678-11683.
  13. Chu, H., et al. (2006). Gamma-Parvin is dispensable for hematopoiesis, leukocyte trafficking, and T-cell-dependent antibody Rresponse. Mol. Cell. Biol. 26:1817-1825. [+SulfoLink]
  14. Lampe, P.D., et al. (2006). Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart. J. Cell Sci. 119:3435-3442. [+SulfoLink]
  15. Nakahara, K., et al. (2006). Maternal ghrelin plays an important role in rat fetal development during pregnancy. Endocrinology 147:1333-1342.
  16. Das, S., et al. (2006). Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins. J. Virol. 80:1191-1203.
  17. Li, X-H, et al. (2004). Estrogen directly induces expression of retinoic acid biosynthetic enzymes, compartmentalized between the epithelium and underlying stromal cells in rat uterus. Endocrinology 145:4756-4762.
  18. Schofield, L., et al. (2002). Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria. Nature 418:785-789.
  19. Quill, T.A., et al. (2001). A voltage-gated ion channel expressed specifically in spermatozoa. Proc. Natl. Acad. Sci. USA. 98:12527-12531.

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



Ordering Information
Bulk quantities: InquireBulk quantities: Inquire    Certificate of AnalysisCertificate of Analysis   Product InstructionsProduct Instructions   MSDSMSDS
Product # Description Pkg. Size Files Price
77605 Imject Maleimide-Activated mcKLH
Formulation: Lyophilized in 10mM sodium phosphate, 150mM sodium chloride, 10mM EDTA, 50mM sucrose, pH 7.2
10mg Product Instructions MSDS for product # 77605 Imject Maleimide-Activated mcKLH $285.00

77606 Imject Maleimide-Activated mcKLH
Formulation: Lyophilized in 10mM sodium phosphate, 150mM sodium chloride, 10mM EDTA, 50mM sucrose, pH 7.2
2mg Product Instructions MSDS for product # 77606 Imject Maleimide-Activated mcKLH $78.00

77610 Imject Maleimide-Activated mcKLH
Formulation: Lyophilized in 10mM sodium phosphate, 150mM sodium chloride, 10mM EDTA, 50mM sucrose, pH 7.2
10 x 10mg Product Instructions MSDS for product # 77610 Imject Maleimide-Activated mcKLH $2,607.00

77663 Imject Maleimide PEGylated mcKLH
Formulation: Lyophilized in 10mM sodium phosphate, 150mM sodium chloride, 10mM EDTA, 50mM sucrose, pH 7.2
10mg Product Instructions MSDS for product # 77663 Imject Maleimide PEGylated mcKLH $300.00

77666 Imject Maleimide-Activated mcKLH Spin Kit
Sufficient for five conjugation reactions.
Kit Contents:
Imject Maleimide-Activated mcKLH, 5 x 2mg
Imject Maleimide Conjugation Buffer, 30mL
Imject Purification Buffer Salts, 5 x 0.84g
Zeba Spin Desalting Columns, 7K MWCO, 5 x 2mL
Kit Product Instructions MSDS for product # 77666 Imject Maleimide-Activated mcKLH Spin Kit $360.00

77164 Imject Maleimide Conjugation Buffer 30mL MSDS for product # 77164 Imject Maleimide Conjugation Buffer $35.00

77611 Imject Maleimide-Activated mcKLH Kit
Sufficient reagents for five hapten-carrier conjugations.
Discontinued product; available until stock is depleted.
Kit Product Instructions MSDS for product # 77611 Imject Maleimide-Activated mcKLH Kit $450.00

77607 Imject Immunogen Kit with Maleimide-Activated mcKLH and BSA
Sufficient reagents for five conjugations with each carrier protein.
Discontinued product; available until stock is depleted.
Kit Product Instructions MSDS for product # 77607 Imject Immunogen Kit with Maleimide-Activated mcKLH and BSA $650.00


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