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crc press - E-Lib FK UWKS

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Cell-Penetrating Peptide Conjugations and Magnetic Cell Labels 335<br />

H 2N<br />

H 2N<br />

H2N<br />

HN<br />

CLIO<br />

HN<br />

O<br />

O<br />

FIGURE 15.4 Synthetic scheme of CLIO–Tat. (From Josephson, L. et al., Bioconjug. Chem.,<br />

10, 186, 1999. With permission.)<br />

extinction coefficient at 343 nm of 8100 M –1 cm –1 . On average, each particle<br />

contains 14 2-pyridyl disulfide groups.<br />

6. To 2.3 mL of 2-pyridyl disulfide derivatized CLIO (16.1 mg/Fe,<br />

288 µmol) was added 1.9 mL of Tat peptide (803 µ M, 1.52 µmol, as<br />

determined by FITC absorbance, extinction coefficient of 73000 M –1 cm –1<br />

at 494 nm in 0.1 M phosphate buffer). The mixture was allowed to react<br />

overnight at room temperature.<br />

7. The solution was applied to a second 20-cm Sephadex G-25 column equilibrated<br />

as above and the excluded volume containing CLIO–Tat was saved.<br />

8. To measure the number of Tat groups attached, the colloid was reacted<br />

with DTT and applied to a microconcentrator as above. Released peptide<br />

in the filtrate was quantitated using FITC fluorescence read against a<br />

standard of FITC. The average number of Tat peptides per CLIO particle<br />

was 4.1 per particle.<br />

NH<br />

O<br />

O<br />

N O<br />

O<br />

S<br />

SPDP<br />

S<br />

S<br />

S<br />

S S<br />

GRKKRRQRRRGYK(Fitc)C-NH 2<br />

GRKKRRQRRRGYK(Fitc)C-NH2<br />

N<br />

N

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