Sulfo-SMCC enzymes with

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. 

The following protocol describes the activation of HRP with sulfo-SMCC. Activation of other enzymes is done similarly, with the appropriate adjustments in the mass of enzyme added to the reaction to account for differences in molecular weight. 

The gel filtration column described in step 3 should be prepared and equilibrated prior to starting the modification reaction. Enzymes preactivated with sulfo-SMCC are available from Thermo Fisher. 

Protocol for the Conjugation of SMCC-Activated Enzymes with Thiolated (Strept)avidin Activation of Enzyme with Sulfo-SMCC... 

Protocols for the activation of enzyme molecules with SMCC (or sulfo-SMCC) can be found in Chapter 20, Section 1.1. Conjugates formed using this method usually result in high-activity complexes giving excellent sensitivity for use in immunoassays or other applications. 

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,... 

The following protocol describes the activation of avidin or streptavidin with sulfo-SMCC and its subsequent conjugation with an enzyme modified to contain sulfhydryls using SATA (Chapter 1, Section 4.1). A method for the opposite approach, wherein the enzyme is activated with SMCC and the avidin component is thiolated, is presented immediately after this protocol. This strategy may be the most common approach to forming these conjugates (Fig. 363). In addition, since there are enzymes commercially available that are preactivated with SMCC (Pierce), their use may be the easiest solution. 

Protocols for the activation of enzyme molecules with SMCC (or sulfo-SMCC) can... 

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