Conjugation-Ready HRP Maleimide

1. Coat antibody or antigen onto the ELISA plate using ICT’s Antibody Coating Buffer or Antigen Coating Buffer., 2. Incubate 8–24 hours at room temperature (RT)., 3. Aspirate the coating solution., 4. Wash each Well twice with ICT’s ELISA Wash Buffer., 5. Block the uncoated regions of the ELISA plate by pipetting 300–400 µL of blocking buffer into each Well. Always use an equal or greater volume of blocking buffer than was used for the coating buffer solution., 6. Incubate 8–24 hours at RT. For best blocking, incubate overnight at RT., 7. Aspirate the blocking buffer; do not wash., 8. Run the assay immediately, or dry the plate for long-term storage and seal in a foil storage bag with a desiccant pack. Store dried and packaged plates at 2-8°C.

Horseradish peroxidase (HRP) enzymes are commonly used as signal reporters in ELISAs, Western blots, and other laboratory techniques. HRP reacts with a substrate (such as our TMB 1-Component HRP MicroWell Substrate, SUBT, catalog 6337) to generate the signal color in the assay that can be detected visually or quantified with a spectrophotometer. To be useful in an assay, the HRP must first be conjugated to a protein or antibody. A stable HRP conjugation linkage is essential to generate reliable results in an assay. To facilitate the conjugation reaction with the antibody or protein, HRP can be linked in advance to maleimide groups, which can then subsequently react with free sulfhydryl groups on the target protein. Our Conjugation-Ready HRP-Maleimide (HRP-M) reagent makes it easy for scientists to create their own detection conjugates. Maleimide conjugation linkers, like HRP-M, are quite stable at pH 6.5-7.5 and specifically react with free sulfhydryl groups (SH) on the target protein, forming a stable thioether linkage. At this pH range of 6.5-7.5, maleimide is only minimally susceptible to hydrolysis; this may be due to the fact that the maleimide ring structure is not directly linked to the aromatic cyclohexane ring. (At more alkaline pH (>8.0), maleimide hydrolyzes at a faster rate into non-reactive maleamic acid. This reduces the efficiency of conjugation as the maleimide functional groups become inactivated prior to coupling to free SH on the antibody or protein.) Following our protocol, the IgG or protein to be labeled must first be treated with Traut’s Reagent (2-Iminothiolane) to introduce free thiols (SH groups) that enable maleimide mediated conjugation. Thiolated protein samples are subsequently incubated with HRP-M, leading to the covalent linkage of HRP to the antibody or protein target. This manual also includes a procedure to determine the ratio of HRP to protein to verify that the conjugation was successful (optional). Older conjugation methods which utilized glutaraldehyde or periodate oxidation to form a Schiff-base have their drawbacks. Glutaraldehyde methods are difficult to control as they often lead to excessive cross-linking and precipitation. Periodate oxidation of the carbohydrate moiety of HRP can be a reasonably successful conjugation method, but this method also has its drawbacks. The oxidation step necessary to form aldehydes on HRP can also in- activate the enzyme, and the Schiff-base linkage must be reduced and stabilized to form a permanent covalent bond between the aldehyde on the HRP and the amino group on the protein. Utilization of HRP-M to facilitate the conjugation reaction avoids HRP enzyme inactivation associated with periodate oxidation procedures, while still controlling the ratio of HRP to the target protein. The 2-step approach described here (thiolation then conjugation) eliminates the risk of uncontrolled cross-linking reactions. These lead to formation of large multimeric protein aggregates and subsequent precipitation events. This is an unavoidable occurrence when using glutaraldehyde techniques. If you have any questions, please visit www.immunochemistry.com or call us at 1-800-824-8540. This product is for research use only. Not for use in diagnostic procedures.