Sulfo-SMCC

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. 

Dissolve 10 mg of a protein or other macromolecule to be activated with sulfo-SMCC in 1 ml of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2. 

Weigh out 2 mg of sulfo-SMCC and add it to the above solution. Mix gently to dissolve. To aid in measuring the exact quantity of crosslinker, a concentrated stock solution may be made in water (or DMSO) and an aliquot equal to 2 mg transferred to the reaction solution. If a stock solution is made, it should be dissolved rapidly and used immediately to prevent extensive hydrolysis of the active ester. As a general guideline of addition for a particular protein activation, the use of a 40- to 80-fold molar excess of crosslinker over the amount of protein present usually results in good activation levels. 

The protocol for using GMBS or sulfo-GMBS in protein-protein crosslinking applications is similar to that of SMCC or sulfo-SMCC (see Section 1.3, this chapter). 

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

A common choice of crosslinker for this type of reaction is sulfo-SMCC, which has been used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for dendrimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic PEG spacer arm to promote dendrimer hydrophilicity after modification. Derivatization of an amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with water-soluble PEG spacers. This modification helps to mask any potential for nonspecific interactions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides for coupling ligands (Figure 7.10). 

Figure 9.62 Sulfo-SMCC can be used to conjugate amine-containing QDs with thiol-containing proteins or other molecules using a two-step coupling procedure.

Add 1 mg of sulfo-SMCC to the QD solution with mixing to dissolve the crosslinker. 

Figure 19.16 A common way of conjugating sulfhydryl-containing haptens to carrier proteins is to activate the carrier with sulfo-SMCC to create an intermediate maleimide derivative. The maleimide groups then can be coupled to thiols to form thioether bonds.

After a carrier protein has been activated with sulfo-SMCC, it is often useful to measure the degree of maleimide incorporation prior to coupling an expensive hapten. Ellman s reagent may be used in an indirect method to assess the level of maleimide activity of sulfo-SMCC-activated proteins and other carriers. First, a sulfhydryl-containing compound such as 2-mercaptoethanol or cysteine is reacted in excess with the activated protein. The amount of unreacted sulfhydryls remaining in solution is then determined using the Ellman s reaction (Chapter 1, Section 4.1). Comparison of the response of the sample to a blank reaction using... 

Figure 19.18 Carrier proteins may be activated with sulfo-SMCC to produce maleimide derivatives reactive with sulfhydryl-containing molecules. The graphs show the gel filtration separation on Sephadex G-25 of male-imide-activated BSA (A) and OVA (B) after reaction with sulfo-SMCC. The first peak is the protein and the second peak is excess crosslinker. The maleimide groups create increased absorbance at 280 nm in the activated proteins.

Dissolve sulfo-SMCC (Thermo Fisher) at a concentration of lOmg/ml in the activation buffer. Immediately transfer the appropriate amount of this crosslinker solution to the vial containing the dissolved carrier protein. 

Note The amount of crosslinker solution to be transferred is dependent on the level of activation desired. Suitable activation levels can be obtained for the following proteins by adding the indicated quantities of the sulfo-SMCC solution. The degree of sulfo-SMCC modification often determines whether the carrier will maintain solubility after activation and coupling to a hapten. Multimeric KLH in particular, is sensitive to the amount of crosslinker addition. KLH usually retains solubility at about 0.1-0.2 times the mass of crosslinker added to BSA. This level of addition still results in excellent activation yields, since KLH is significantly larger than most of the other protein carriers. 

Add the following quantities of sulfo-SMCC solution to each ml of carrier protein solution ... 

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. 

The gel filtration column described in step 3 should be prepared and equilibrated prior to starting the modification reaction. Enzymes preactivated with sulfo-SMCC are available from Thermo Fisher. 

Add 6mg of sulfo-SMCC (Thermo Fisher) to the HRP solution. Mix to dissolve and react for 30 minutes at room temperature. Alternatively, two 3 mg additions of crosslinker may be done—the second one after 15 minutes of incubation—to obtain even more efficient modification. 

Figure 21.13 Sulfo-SMCC may be used to activate antibody molecules for coupling to thiolated toxin components. An intact A-B toxin molecule can be modified to contain sulfhydryls by treatment with 2-iminothiolane. Thiolation with this reagent retains the cytotoxic properties of the toxin while generating a sulfhydryl for conjugation. Reaction of the thiolated toxin with the maleimide-activated antibody creates the immunotoxin through thioether bond formation.

Add 1.0 mg of sulfo-SMCC (Thermo Fisher) to each ml of (strept)avidin solution. Mix to dissolve. 

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

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