Dendrimer epoxy activation

Dilute the reaction mixture 10-fold with 50 percent methanol/water and purify the epoxy-activated dendrimer by repeated ultrafiltration using a membrane with a molecular... 

The epoxy-activated dendrimer may be conjugated to thiol-containing proteins by reaction in 50 mM sodium phosphate, pH 7.2. The reaction can be done at 4°C or at room temperature for 8-16 hours to form thioether linkages. 

Other than the epoxy groups available on one Priostar dendrimer type and a methyl ester available on a PAMAM dendrimer, the commercial suppliers generally don t offer a selection of spontaneously reactive dendrimers for bioconjugation purposes. For this reason, most of the applications published for coupling biomolecules to dendrimers have used various modification or activation steps to create the appropriate reactive groups for conjugation (e.g., Leon etal., 1996). 

Figure 7.18 Amine-containing dendrimers can be activated with epibromohydrin to result in the formation of reactive epoxy groups on the dendrimer surface. This reactive intermediate then can be used to conjugate with thiol-containing proteins, such as thiolated alkaline phosphatase. The reaction results in the formation of a thioether bond.

The surfaces included PLL, polystyrene, epoxy-terminated polyethylene glycol (PEG) or dendrimer slides, various amine-derivatized surfaces, and nitrocellulose-coafed slides. All proteins were printed in PBS, rinsed in TBS, and then blocked in 3% nonfat dry milk powder dissolved in TBS-0.1% Tween-20. A final rinse in TBS was performed prior to incubation. While no attempt was made to optimize print buffer or blocking conditions for each of the selected surfaces, if was apparenf fhaf wifh fhe exception of activated polystyrene, most chemistries performed af abouf fhe same levels, i.e., within two- to threefold af saturation. 

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