Sulfo-SMCC carrier proteins

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... 

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. 

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.

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 ... 

After a carrier protein has been activated with sulfo-SMCC it is often useful to... 

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