Avidin thiolation

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

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. 

Immediately mix the thiolated enzyme with an amount of maleimide-activated (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. 

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. 

Conjugation of SMCC-Activated Enzyme with Thiolated (Strept)avidin... 

Ideally, thiolated avidin will be coupled immediately to G4-SMCC, although it can be kept for a few weeks at -80°C. If thawed, check its SH content as described above... 

Antibody, enzyme, peptide, protein, or thiolated oligonucleotide to be conjugated to avidin. 

Purify the thiolated avidin on a 7 x 250 mm size exclusion column, such as Sephadex G-25 equilibrated in 0.1 M phosphate buffer. 

Equation (1) allows the determination of the average number of moles of enzyme or antibody that can be conjugated with each of avidin (see Note 6). For a 1 1 protein-avidin conjugate, avidin should be modified with 1.2-1.5 thiols/mol. Thiolated avidin prepared by the above procedure can be stored in the presence of 2 voM sodium azide at 4°C for 4-6 wk... 

In this step, which should be completed prior to the deprotection of thiolated avidin, some of the ammo groups from the antibody or enzyme are transformed into maleimide groups by reacting with a bifunctional crosslinker, SMCC (see Note 7). 

This procedure is carried out immediately before reacting thiolated avidin with the maleimide derivative of the antibody or enzyme prepared in Section 3.1.2. 

Add 11 pL of TCEP solution to the thiolated avidin solution Incubate for 15 min at room temperature... 

Add the thiolated avidin-TCEP mixture dropwise to the dialyzed maleimide-derivatized protein solution with stirring Continue stirring for 1 h at room temperature, followed by stirring overnight at 4°C. 

It is essential that the procedure described in Section 3.1.2. be performed approx 24 h before the procedure described in Section 3.1 3, because the deprotected thiolated avidin and the maleimide derivative of the protein are unstable. Purification of the maleimide-derivatized protein by size exclusion chromatography can be performed more rapidly than dialysis, however, the former leads to dilution of the protein and a decrease in the yield of the conjugate. 

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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