Molar excess limiting quantities

Since the active ester end of the molecule is subject to hydrolysis (half-life of about 20 minutes in phosphate buffer at room temperature conditions), it should be coupled to an amine-containing protein or other molecule before the photolysis reaction is done. During the initial coupling procedure, the solutions should be protected from light to avoid decomposition of the phenyl azide group. The degree of derivatization should be limited to no more than a 5- to 20-fold molar excess of sulfo-SBED over the quantity of protein present to prevent possible precipitation of the modified molecules. For a particular protein, studies may have to be done to determine the optimal level of modification. 

Add a quantity of the crosslinker solution to the protein solution to provide a 1- to 10-fold molar excess of reagent over the concentration of protein. The use of lower molar ratios will limit the potential for oligomerization of proteins in solution. A series of reactions using different concentrations of crosslinker may have to be done to determine the optimal level to use for a particular application. 

With mixing, add an aliquot of the biotin solution to the protein solution to obtain at least a 10-fold molar excess over the quantity of protein present. As thiols typically are present in limiting amounts on proteins, the use of a high-molar reagent ratio is not required to achieve acceptable yields of biotinylation. 

---