7. It is recommended before cleavage to analyze the peptide
sequence and use an appropriate cleavage cocktail accordingly
to prevent unwanted side reactions while the cleavage step.
8. DMSO should be avoided with peptides that contains Cys,
Trp, or Met as they can easily undergo oxidation.
9. An alternative method for purifying a labeled peptide is using
desalting columns with an appropriate molecular weight cutoff
(MWCO). This will allow to remove the fluorophore, that has a
lower molecular weight, from the peptide without the need of a
HPLC purification.
10. Once the fluorophore conjugated peptide has been purified
and dissolved It is possible to determine its concentration
using the Lambert–Beer law knowing the extinction coefficient
and the absorption/emission wavelength of the fluorophore
(usually provided by the manufacturer) with a UV
spectrophotometer.
A ¼ ε  l  c ! c ¼
A
εÂl :
where ε: molar absorptivity (L mol
À1 cm
À1 ), l: cuvette length
(cm), c concentration (mol L
À1 ), A: absorbance.
11. To synthesize an asymmetrical peptide on-resin, load the resin
with a 50/50 mixture of Fmoc-Asp(OtBu)-OH and FmocVal-OH on a 2-chlorotrityl chloride resin as the initial step
using diisopropylethylamine (DIPEA) (resin–amino acid–
DIPEA in 1:4:8) in DCM for 30 min followed by capping
with methanol (DCM–MeOH–DIPEA 17:2:1). Then follow
the conventional peptide synthesis explained Subheading 3.1
and the addition of PEG-diacid.
12. Linker length optimization is going to be crucial to optimize
the dimeric binder candidate for further studies. We recommend to test different PEG n diacids with different lengths and
screen for the best candidate.
13. The dilution curve should be planned so that the expected K d
concentration is in the middle of the curve. If the binding
constant is unknown, it will need several optimization steps
to find the perfect range.
14. To prevent light quenching the fluorescent probe and to assure
its quality overtime we recommend covering it always with
aluminum foil and preparing all the solutions with the probe
in ice.
15. We strongly recommend filtrating all the buffers for ITC and if
possible the same for the peptide and protein solutions to have
accurate results.
16. The biotinylated peptides can be synthesized following the
standard solid-phase Fmoc synthesis procedure explained in
Subheading 3.1. The biotin can be coupled using the same
PDZ Domain Peptide Inhibitors
175
sequence and use an appropriate cleavage cocktail accordingly
to prevent unwanted side reactions while the cleavage step.
8. DMSO should be avoided with peptides that contains Cys,
Trp, or Met as they can easily undergo oxidation.
9. An alternative method for purifying a labeled peptide is using
desalting columns with an appropriate molecular weight cutoff
(MWCO). This will allow to remove the fluorophore, that has a
lower molecular weight, from the peptide without the need of a
HPLC purification.
10. Once the fluorophore conjugated peptide has been purified
and dissolved It is possible to determine its concentration
using the Lambert–Beer law knowing the extinction coefficient
and the absorption/emission wavelength of the fluorophore
(usually provided by the manufacturer) with a UV
spectrophotometer.
A ¼ ε  l  c ! c ¼
A
εÂl :
where ε: molar absorptivity (L mol
À1 cm
À1 ), l: cuvette length
(cm), c concentration (mol L
À1 ), A: absorbance.
11. To synthesize an asymmetrical peptide on-resin, load the resin
with a 50/50 mixture of Fmoc-Asp(OtBu)-OH and FmocVal-OH on a 2-chlorotrityl chloride resin as the initial step
using diisopropylethylamine (DIPEA) (resin–amino acid–
DIPEA in 1:4:8) in DCM for 30 min followed by capping
with methanol (DCM–MeOH–DIPEA 17:2:1). Then follow
the conventional peptide synthesis explained Subheading 3.1
and the addition of PEG-diacid.
12. Linker length optimization is going to be crucial to optimize
the dimeric binder candidate for further studies. We recommend to test different PEG n diacids with different lengths and
screen for the best candidate.
13. The dilution curve should be planned so that the expected K d
concentration is in the middle of the curve. If the binding
constant is unknown, it will need several optimization steps
to find the perfect range.
14. To prevent light quenching the fluorescent probe and to assure
its quality overtime we recommend covering it always with
aluminum foil and preparing all the solutions with the probe
in ice.
15. We strongly recommend filtrating all the buffers for ITC and if
possible the same for the peptide and protein solutions to have
accurate results.
16. The biotinylated peptides can be synthesized following the
standard solid-phase Fmoc synthesis procedure explained in
Subheading 3.1. The biotin can be coupled using the same
PDZ Domain Peptide Inhibitors
175