Antibodies SMCC-activated enzymes

Figure 20.5 Antibodies may be modified with 2-iminothiolane at their amine groups to create sulfhydryls for conjugation with SMCC-activated enzymes. The maleimide groups on the derivatized enzyme react with the thiols on the antibody to form thioether bonds.

Dissolve the enzyme to a concentration of 1 - 2 mg/ml in Soln. A (if it is delivered in another buffer, dialyze against Soln. A). Mix 1 mg -galactosidase in Soln. A with 0.25 mg SMCC activated antibody (concentration about 1 mg/ml for activation see Protocol 4.1.3). Shake at RT for 2 h, dialyze or desalt on a Sephadex G-25 column against Soln. B and concentrate to about 2 mg/ml. Mix 9 vol. of the concentrated conjugate with 1 vol. Soln. C and store without further purification at 4 °C. 

One note should be mentioned before proceeding when conjugating antibody molecules with (B-galactosidase, the antibody usually is activated with sulfo-SMCC first to take advantage of the indigenous sulfhydryl groups on the enzyme. Therefore, if (3-gal is being used, substitute the antibody for the enzyme mentioned in this protocol,... 

SMCC frequently is used to prepare hapten-carrier or antibody-enzyme conjugates. In both applications, one of the molecules is activated (usually the carrier or the enzyme) with the... 

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. 

Figure 20.3 The reaction of SMCC with the amine groups on enzyme molecules yields a maleimide-activated derivative capable of coupling with sulfhydryl-containing antibody molecules.

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