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Amylose Resin Assay of MBP-5HIS to QDs. Affinity columns (25 mm 76 mm) of amylose resin (New England Biolabs, Beverly, MA) were setup and equilibrated with 10 mM Na-tetraborate buffer (pH 9.55).9 Then, 100 pmol of CdSe-ZnS QDs capped with 6, 10, and a 1:1 mixture of 6 and 10 was mixed with the equivalent of 20 maltose binding proteins appended with an oligohistidine tail (MBPHIS) per QD in 200 íL aliquots of 10 mM borate buffer at pH 9.55 and allowed to self-assemble for 1 h.9,16 Duplicates of each sample tested were loaded onto the amylose resin pre-equilibrated in buffer. After binding to the column, samples were washed with 1 mL of buffer, eluted with 20 mM maltose in buffer, and the eluant captured. The eluant was brought up to 500 íL with 20 mM maltose in buffer. A quantity of 100 íL of all samples was then loaded onto a microtiter well plate in triplicate, and the PL was measured on a Tecan Safire plate reader (Tecan US, Research Triangle Park NC). By normalizing the PL of the DHLA-capped QD self-assembly mixture to 100%, we could estimate the percent of each QD sample binding to the column and being released with maltose relative to the solution containing DHLA-capped QDs (Figure 4).  untitled.gif |
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