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Peroxidase (HRP) Labeling Reagent, Thiol Reactive

 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).

Reaction of thiol compounds and maleimides

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.

HRP-labelling of an antibody with H1621

Figure 2. HRP-labelling of an antibody with H1621

Product

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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).

Activity of HRP-labeled Antibody

Figure 3. Activity of HRP-labeled Antibody

Goat Anti-Mouse IgG was labeled with the HRP using H1621
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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