A conjugate reaction

Purify the thiolated protein from excess DTT by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, ImM EDTA, pH 7.2. The modified protein should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

Imidoester crosslinkers are highly water-soluble, but undergo continuous degradation due to hydrolysis. The half-life of the imidate functionality is typically less than 30 minutes, especially in the alkaline conditions of the reaction medium (Hunter and Ludwig, 1962 Browne and Kent, 1975). Concentrated stock solutions may be prepared before addition of a small amount to a conjugation reaction, but they should be dissolved rapidly and used immediately. 

SMPB contains a hydrophobic cross-bridge and relatively nonpolar ends, which allows the reagent to permeate membrane structures. Due to its water-insolubility, it must be dissolved in an organic solvent prior to adding an aliquot to a reaction mixture. The solvents DMF and DMSO work well for this purpose. A concentrated stock solution prepared in these solvents allows for easy addition of a small amount to a conjugation reaction. Long-term storage in these solvents is not recommended due to slow water pickup and possible hydrolysis of the NHS ester end. 

Cascade Blue acetyl azide is soluble in aqueous solution, but the reactive azide group will hydrolyze and should be used immediately in a conjugation reaction. A concentrated stock solution may be prepared in water, dissolved quickly, and an aliquot quickly added to a buffered reaction medium. For aqueous reactions, a pH range of 7-9 is optimal. Avoid amine-containing buffers. 

Although the formation of hippuric acid is a conjugation reaction, many metabolic conversions require the ability of living organisms to oxidize compounds and in... 

Heterobifunctional cross-linking reagents also may be used to site-direct a conjugation reaction toward particular parts of target molecules. Amines may be coupled on one molecule while sulfhydryls or carbohydrates are targeted on another molecule. Directed coupling often is important in preserving critical epitopes or active sites within macromolecules. For instance, antibodies may be coupled to other proteins... 

It is common knowledge that in the case of chemical induction, the primary reaction produces useful work for a conjugated reaction to proceed. As two chemical reactions are conjugated, they must both be rigidly connected to one another, because successful realization of this scheme permanently demands useful work to be produced by the primary reaction. Termination of active site generation in the primary reaction leads to secondary reaction termination. 

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