Figure 6. Conjugation of mSiNCs to Tf.... | Rockefeller University Press

$Figure 6. Conjugation of mSiNCs to Tf. (A and B) The scheme for conjugating mSiNC to Tf, using (N-ε-maleimidocaproyloxy) sulfosuccinimide ester (sulfo-EMCS). (C, top) Superdex 200 gel filtration HPLC chromatograms for the samples obtained at 0 and 60 min after the mSiNC-Tf conjugation reaction was initiated. Peak 1, maleimide-Tf dimers (but not those cross-linked by mSiNC). Peak 2, overlapped eluent of mSiNC-Tf and maleimide-Tf. Peak 3, unconjugated mSiNC. The explanation for these assignments is given in the text. (bottom) Superdex 200 elution pattern of Tf monomers, dimers, and oligomers, generated by chemical cross-linking of Tf with bis(sulfosuccinimidyl)suberate. (D) Superdex 75 gel filtration HPLC chromatograms for the samples obtained at 0, 30, and 60 min after the mSiNC-Tf conjugation reaction was initiated. Peak 1, mSiNC-conjugated Tf (mSiNC-Tf). Peak 2, maleimide-Tf. Peak 3, unconjugated mSiNCs. The explanation for these assignments is given in the text. Additionally, these chromatograms indicate that about half of the mSiNCs added for this conjugation reaction became conjugated to maleimide-Tf in 60 min. Note that all of these peaks occur after the void volume determined by Blue Dextran 2000. Data in early time periods were deleted in C and D because no eluent peaks were detectable during these early periods. (E) Distributions of fluorescence intensities of individual mSiNC or mSiNC-Tf spots (measured in 530 × 530–nm areas) after the conjugation reactions for 0, 30, and 60 min. Excitation was by the 405-nm line of a solid-state laser. A single log-normal function (red curves) gave a good fit, and the two-component fitting did not significantly improve the fit, but nevertheless, the latter gave an estimate of the possible dimer fraction of less than a few percentages. Typical results among 7 (C, top), 3 (C, bottom), 11 (D), and 6 (E) entirely independent experiments are shown. a.u., arbitrary unit.$

Figure 6.

Conjugation of mSiNCs to Tf. (A and B) The scheme for conjugating mSiNC to Tf, using (N-ε-maleimidocaproyloxy) sulfosuccinimide ester (sulfo-EMCS). (C, top) Superdex 200 gel filtration HPLC chromatograms for the samples obtained at 0 and 60 min after the mSiNC-Tf conjugation reaction was initiated. Peak 1, maleimide-Tf dimers (but not those cross-linked by mSiNC). Peak 2, overlapped eluent of mSiNC-Tf and maleimide-Tf. Peak 3, unconjugated mSiNC. The explanation for these assignments is given in the text. (bottom) Superdex 200 elution pattern of Tf monomers, dimers, and oligomers, generated by chemical cross-linking of Tf with bis(sulfosuccinimidyl)suberate. (D) Superdex 75 gel filtration HPLC chromatograms for the samples obtained at 0, 30, and 60 min after the mSiNC-Tf conjugation reaction was initiated. Peak 1, mSiNC-conjugated Tf (mSiNC-Tf). Peak 2, maleimide-Tf. Peak 3, unconjugated mSiNCs. The explanation for these assignments is given in the text. Additionally, these chromatograms indicate that about half of the mSiNCs added for this conjugation reaction became conjugated to maleimide-Tf in 60 min. Note that all of these peaks occur after the void volume determined by Blue Dextran 2000. Data in early time periods were deleted in C and D because no eluent peaks were detectable during these early periods. (E) Distributions of fluorescence intensities of individual mSiNC or mSiNC-Tf spots (measured in 530 × 530–nm areas) after the conjugation reactions for 0, 30, and 60 min. Excitation was by the 405-nm line of a solid-state laser. A single log-normal function (red curves) gave a good fit, and the two-component fitting did not significantly improve the fit, but nevertheless, the latter gave an estimate of the possible dimer fraction of less than a few percentages. Typical results among 7 (C, top), 3 (C, bottom), 11 (D), and 6 (E) entirely independent experiments are shown. a.u., arbitrary unit.

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