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Thermo Scientific Imject Maleimide-Activated cBSA is cationized bovine serum albumin formulated for conjugation to sulfhydryl-containing peptides and haptens for use in antibody production procedures.
Purified BSA has been cationized by chemically capping carboxyl side-chains with primary amines and then modified with SMCC crosslinker to attach maleimide groups capable of forming covalent crosslinks with sulfhydryl (-SH) moieties on cysteine residues of peptides and other molecules. Immunogens are easily prepared from peptide antigens that have been engineered with a terminal cysteine residue as a precise conjugation point. Several antigen molecules can be attached per carrier protein molecule without blocking intended epitopes by crosslinking to primary amines or carboxylates within the peptide sequence.
- Maleimide-activated cationized BSA, supplied lyophilized in PBS (pH) with stabilizer
- Greater than four (4) functional maleimide groups per cBSA molecule
- Ideal for conjugating cysteine-containing peptide antigens to make immunogen
- Conjugation reaction complete in 2 hours; results in stable, covalent thioether bonds
- Single polypeptide protein with molecular mass > 67,000 Daltons
- Contains 59 lysine residues, plus numerous other primary amines added by cationization
- Net positive charge (pI >11) confers solubility in conditions not suitable for the native BSA (pI 5.1)
- Significantly greater immunogenicity than native BSA because the positively charged protein is processed differently by the immune-response system.
|Summary of carrier protein activation and conjugation to cysteine-containing peptide antigen. Maleimide-Activated cBSA is supplied lyophilized in the form shown at the center of the diagram. 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 maleimide activations.
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 adequate conjugation with the carrier can be achieved.
Imject cBSA is prepared by modifying native BSA with excess ethylenediamine, essentially capping all negatively-charged carboxyl groups with positively-charged primary amines. The result is a highly positively-charged protein (pI > 11) that has significantly increased immunogenicity compared to native BSA. Imject cBSA (previously called SuperCarrier) is an excellent choice for immunogen preparation when greater immunogenicity is needed relative to BSA but conditions are not amenable to using keyhole limpet hemocyanin (KLH) or Blue Carrier Protein (CCH).
- Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Chapter 5 discusses the use of carrier proteins.
- Hermanson, G.T. (2008). Bioconjugate Techniques. 2nd edition, Academic Press, New York. (Part No. 20036). Chapter 19 discusses carrier protein uses and the maleimide-activation chemistry.
- Muckerheide, et al. (1987). Cationization of protein antigens. I. Alteration of immunogenic properties. J. Immunol. 138, 833-837.
- Muckerheide, et al. (1987). Cationization of protein antigens. II. Alteration of regulatory properties. J. Immunol. 138, 2800-2804.
- Domen, P.L., et al. (1987). Cationization of protein antigens. III. Abrogation of oral tolerance. J. Immunol. 139, 3195-3198.
- Apple, R.J., et al. (1988). Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells. J. Immunol. 140, 3290-3295.
- Anderson, J.P., et al. (2006). Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic lewy body disease. J. Biol. Chem. 281:29739-29752.
- Smith, E.R., et al. (2005). A human protein complex homologous to the Drosophila MSL complex is responsible for the majority of histone H4 acetylation at lysine 16. Mol. Cell. Biol. 25:9175-9188.
Review of antibody production and immunogen preparation methods
Review of sulfhydryl-reactive (maleimide) crosslinker chemistry
Carrier protein activation and conjugation data for immunogen preparation
Maleimide-Activated Blue Carrier Protein and Conjugation Kit
Maleimide-Activated KLH and Conjugation Kit
Maleimide-Activated BSA and Conjugation Kit
Cationized BSA (non-activated)