SMCC-Activated Enzymes

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. 

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

Hydrazide functional groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can specifically bind to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

Other molecules can be used in this type of assay approach. Hydrazide-modified avidin, streptavidin, lectins, biocytin, fluorescent probes, and other detectable molecules can specifically detect glycoconjugates in this manner (Wilchek and Bayer, 1987). 

The activation of enzymes using adipic acid dihydrazide and EDC is identical to the procedure outlined for the modification of avidin or streptavidin (Chapter 13, Section 5). 

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SMCC-activated enzyme containing sulfhydryl-reactive maleimide groups... 

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.

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

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. 

SMCC-Activated Enzyme Containing Sulfhydryl-Reactive Maleimide Groups... 

Conjugation of SMCC-Activated Enzyme with Thiolated Avidin... 

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. 

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. 

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. 

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. 

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