NHS Ester-Maleimide Heterobifunctional Crosslinker-Mediated Hapten-Carrier Conjugation

NHS Ester-Maleimide Heterobifunctional Crosslinker-Mediated Hapten-Carrier Conjugation 

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 

Dissolve the carrier of choice at a concentration of lOmg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2-7. 5 (activation buffer). 

Note For use of native, multi-subunit KLH, increase the NaCl concentration to 0.9 M. 

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. 

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