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Malemide groups are known to be able to specifically react with free thiol (SH, sulfhydryl) groups under biological conditions in the pH range of 6.5–7.5 to form a stable thioether moiety (Figure 1).
Figure 1. Reaction of thiol compounds and maleimides
Horseradish Peroxidase Maleimide Conjugate [H1621] features:
• The thiol-reactive maleimide group of H1621 can be used for the conjugation to proteins and peptides containing free thiols.
• For example, H1621 enables you to easily do the HRP-labelling of an antibody (Figure 2).
• Packaged for single use purposes and thus do not require weighing prior to use.
Figure 2. HRP-labelling of an antibody with H1621
Product
Application : HRP-labelling of an antibody with H1621
In case of antibodies without free thiol (SH) groups, disulfide moieties in proteins can be reduced by reductants such as DTT [D3647] or 2-MEA [A0296] to reveal free thiols. Furthermore, thiol groups can be introduced to primary amines by adding SATA [S0431], SATP [S0859] or 2-Iminothiolane.
Here we introduce a method that reduce native disulfide bonds in the Goat Anti-Mouse IgG, and are labeled with the HRP using H1621. For more information, see the product detail page of H1621.
Protocol
1) Add DTT to a final concentration equal to 3 mole equivalents per mole equivalent of antibody present.
2) Incubate for 90 minutes at 37 °C.
3) Purify the reduced IgG by gel filtration or ultrafiltration, dialysis.
4) Add equal amount of H1621 (by weight) to a purified antibody and Incubate for 2 hours at room temperature (25 °C).
Figure 3. Activity of HRP-labeled Antibody
was analysed by ELISA using a Mouse IgG coated plate.
Mouse IgG could be detected sufficiently even if the labeled antibody was diluted 5 ng/mL or more.
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Anti-HRP Antibody (*This antibody is unavailable in the U.S. and China.)