Conjugation enzymes using SMCC

The following protocol should be compared to the method described for SATA thiolation in Chapter 1, Section 4.1. Although the procedures are slightly dissimilar, the differences indicate the flexibility inherent in the chemistry. For convenience, the buffer composition indicated here was chosen to be consistent throughout this section on enzyme-antibody conjugation using SMCC. Other buffers and alternate protocols can be found in the literature. 

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

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. 

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. 

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

Immediately mix the SMCC-activated enzyme with an amount of thiolated (strept)avidin to obtain the desired molar ratio of enzyme-to-(strept)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. 

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. 

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