Thermo Scientific Imject Maleimide-Activated BSA products enable conjugation of sulfhydryl-containing peptide antigens to immunogenic bovine serum albumin carrier protein for immunization and antibody production.
Purified, carrier-grade BSA 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. Several antigen molecules can be conjugated to each BSA molecule to increase their immunogenicity and likelihood of eliciting immunized animals to produce high-titer antisera to the epitopes of interest. Select from among several package sizes of activated BSA protein, or choose the complete conjugation kit that includes optimized buffers and trouble-free spin desalting columns.
- Convenient kit – containing stabilized, lyophilized Maleimide-Activated BSA (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 15 to 20 moles of sulhydryl-reactive maleimide per mole of BSA, 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 – pure BSA (Fraction V) is activated using high-quality Thermo Scientific Sulfo-SMCC crosslinker and formulated to ensure high stability and consistent performance
- Immunogenic and soluble – moderately immunogenic and highly soluble, BSA is useful for preparation of the primary immunogen or as an irrelevant hapten-carrier for antibody screening and immunoassays after using KLH as the carrier protein to generate the immune response against the hapten
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.
Bovine serum albumin (BSA) is a convenient protein for a variety of uses in the laboratory because, like most abundant plasma proteins, it is very stable and soluble. Numerous carboxyl groups account for the net negative charge (pI 5.1) of BSA and its high solubility (compare to positively-charged, cationized BSA). In addition, the 67kDa protein is sufficiently large and complex to be fully immunogenic. Consequently, BSA is a popular carrier protein for conjugation to haptens and other weak antigens to make them more immunogenic for the purpose of antibody production. If not used to prepare the primary immunogen, BSA is a useful carrier protein to make antigens more amenable to analysis (e.g., screening assays involving plate coating) of antibodies that were produced using KLH or another more immunogenic carrier protein.
|Summary of BSA carrier protein activation and conjugation to peptide antigen. Maleimide-Activated BSA 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.
- 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.
- Karim, A.S. (1995). Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester. Nucl. Acid. Res. 23(11), 2037-2040.
- Gembitsky, D.S., et al. (2004). A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation. Mol. Cell. Proteomics 3:1102-1118.
- Walker, C., et al. (2006). Mortalin-based cytoplasmic sequestration of p53 in a nonmammalian cancer model. Am. J. Pathol. 168:1526-1530.
Review of antibody production and immunogen preparation methods
Review of sulfhydryl-reactive (maleimide) crosslinker chemistry
Carrier protein activation and conjugation data for immunogen preparation
Sulfo-SMCC – crosslinker for activating BSA amines to make them sulfhydryl-reactive
SulfoLink Immobilization Kit for Peptides – for preparing an affinity column with a cysteine peptide
Bovine Serum Albumin (non-activated BSA) – for hapten conjugation through amines or carboxylates
Keyhole Limpet Hemocyanin (KLH) and other Carrier Proteins