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Cell-Penetrating Peptide Conjugations and Magnetic Cell Labels 335
H 2N
H 2N
H2N
HN
CLIO
HN
O
O
FIGURE 15.4 Synthetic scheme of CLIO–Tat. (From Josephson, L. et al., Bioconjug. Chem.,
10, 186, 1999. With permission.)
extinction coefficient at 343 nm of 8100 M –1 cm –1 . On average, each particle
contains 14 2-pyridyl disulfide groups.
6. To 2.3 mL of 2-pyridyl disulfide derivatized CLIO (16.1 mg/Fe,
288 µmol) was added 1.9 mL of Tat peptide (803 µ M, 1.52 µmol, as
determined by FITC absorbance, extinction coefficient of 73000 M –1 cm –1
at 494 nm in 0.1 M phosphate buffer). The mixture was allowed to react
overnight at room temperature.
7. The solution was applied to a second 20-cm Sephadex G-25 column equilibrated
as above and the excluded volume containing CLIO–Tat was saved.
8. To measure the number of Tat groups attached, the colloid was reacted
with DTT and applied to a microconcentrator as above. Released peptide
in the filtrate was quantitated using FITC fluorescence read against a
standard of FITC. The average number of Tat peptides per CLIO particle
was 4.1 per particle.
NH
O
O
N O
O
S
SPDP
S
S
S
S S
GRKKRRQRRRGYK(Fitc)C-NH 2
GRKKRRQRRRGYK(Fitc)C-NH2
N
N