Thermo Scientific Pierce Maleimide Activated Horseradish Peroxidase and Kit is for preparation of HRP conjugates with proteins, peptides or other ligands that contain sulfhydryl groups, such as reduced cysteines.
This product consists of horseradish peroxidase (HRP) that has been modified with Sulfo-SMCC (Part No. 22322) to attach several maleimide groups per HRP molecule while retaining the peroxidase activity. The activated HRP will covalently attach to proteins or other molecule containing sulfhydryl groups (e.g., cysteines). HRP-conjugates of antibodies, proteins, peptides and other thiol-containing reporter probes are easily made using this method. The complete kit includes the activated HRP as well as two types of reagents for preparing sulfhydryl-ready antibodies (IgG) or proteins for conjugation.
- Activated HRP – horseradish peroxidase (HRP) modified with maleimide groups for conjugation to sulfhydryl molecules
- Sulfhydryl-reactive – maleimide groups conjugate to reduced thiols (-SH), as in the side-chain of cysteine residues
- High activity HRP – enzyme activity is greater than 240 units/mg; lyophilized, activated enzyme is stable for at least 12 months at 4°C
- Complete kit – includes the activated HRP as well as two types of reagents for sulfhydryl-ready antibodies (IgG) or proteins for conjugation
Reaction scheme for conjugation of Maleimide Activated HRP to a sulfhydryl. In this diagram, (R) represents the maleimide-activated peroxidase; (P) represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH). For more information, see our review of Maleimide Reaction Chemistry.
The complete kit for Maleimide Activated Horseradish Peroxidase contains reagents for exposing or added the necessary sulfhydryl groups on antibodies (IgG) or practically any other protein. These general strategies are described briefly in the applications section of our review of Maleimide Reaction Chemistry. Of course, any protein that contains cysteines has sulfhydryl groups (-SH), but they must be reduced (not in the form of disulfide bonds) to be conjugated. Antibodies also contain disulfide bonds that can be targeted as antibody labeling sites; the hinge-region disulfide bonds in IgG can be selectively cleaved with the mild reducing agent 2-Mercaptoethylamine (Part No. 20408), which is included in the complete kit. Alternatively, sulfhydryl groups can be added to proteins (or any amine-containing molecule) using SATA reagent (Part No. 26102), which also is included in the kit.
- Hashida, S., et al. (1984). More useful maleimide compounds for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge. J. Appl. Biochem. 6, 56-63.
- Imagawa, M., et al.(1982). Characteristics and evaluation of antibody-horseradish peroxidase conjugates prepared by using a maleimide compound, glutaraldehyde, and periodate. J. Appl. Biochem. 4, 41-57.
- O’Sullivan, M.J., et al. (1979). Comparison of two methods of preparing enzyme-antibody conjugates: application of these conjugates for enzyme immunoassay. Anal. Biochem. 100, 100-108.
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