Streptavidin enzymes using SMCC

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. 

If p-galactosidasc is used to conjugate with an SMCC-activated avidin or streptavidin, then there is no need to thiolate the enzyme, since it contains sulfhydryls in its native state (Sivakoff and Janes, 1988 Fujiwara et al., 1988). For conjugations using horseradish peroxidase, alkaline phosphatase, or glucose oxidase, however, thiolation is necessary to add the requisite sulfhydryls. 

A variation of the above method can be used, wherein the enzyme is first activated with SMCC and conjugated to a thiolated avidin or streptavidin molecule. This approach probably is the most common way of preparing avidin—enzyme conjugates, and since the preactivated enzymes are readily available (Pierce), it also may be the easiest. 

Immediately mix the SMCC-activated enzyme with an amount of thiolated avidin (or streptavidin) to obtain the desired molar ratio of enzyme to avidin in the conjugate. Use of a 4 1 (enzyme-.avidin) molar ratio in the conjugation reaction usually results in high-activity conjugates suitable for use in many enzyme-linked immunoassay procedures employing the LAB approach. 

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See also in sourсe #XX -- [ ]

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