crc press - E-Lib FK UWKS

Quantification of Cell-Penetrating Peptides and Their Cargoes 265
O
S
(CH 2 ) 4
O
HO
C
R
O
(a)
(c)
H
N
FIGURE 12.1 Structures of three reporter groups used in studies of transportan. (a) Biotinyl-,
(b) 2-amino benzoyl (or Abz, λ ex: 320 nm, λ em: 420 nm), and (c) the fluorescein group (λ ex:
494 nm, λ em: 520 nm). The R denotes the attachment of the label to the primary amino group
on the CPP.
of the label (NBD-group, for example) on CPPs that readily flow out from cells,
e.g., MAP and transportan, could yield results biased toward lower uptake.
Scheller et al. discovered a wash-out effect connected to amphipaticity of the
MAPs. 8 Analogues with lower hydrophobic moment had a lower resistance to washout.
The higher ability of amphipathic peptides to associate with intracellular structures
is suggested to be the main contributor to this phenomenon. Methods reviewed in this
chapter on quantification of cell-penetrating peptides and their cargoes are as follows:
• Radiolabeling*
• Biotinylation*/Cell-ELISA
• Fluorescence microscopy/Spectrophotometer/FACS
• HPLC detection
• Immunodetection
• Detection of enzyme activity
• Fluorogenic construct assay*
NH
* Practical examples of the method follow the text.
H
H
OH
C O
O
C
R
O
NH 2
O
(b)
N
H
R