NHS Ester—Maleimide-Mediated Conjugation Protocols

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

Dissolve (strept)avidin (Thermo Fisher) in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of lOmg/ml. 

0 mg of sulfo-SMCC (Thermo Fisher) to each ml of (strept)avidin solution. Mix to dissolve. 

React for 30-60minutes at room temperature. Since maleimide groups are labile in aqueous solution, extended reaction times should be avoided. 

Immediately purify the maleimide-activated (strept)avidin away from excess crosslinker and reaction by-products by gel filtration on a desalting resin. A spin column will facilitate the 

Heterobifunctional cross-linking agents can be used to control the degree of protein conjugation, thus limiting polymerization and controlling the molar ratio of each component in the final complex (Chapter 5). Particularly useful heterobifunctionals 

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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