should have their N-terminus acetylated to mimic an internal
protein sequence by avoiding an electrostatic dipole contribution (see [15] for additional details). In addition, the
C-terminus should be amidated to neutralize the free carboxylate group.
4. Peptides are typically purified via high-performance liquid
chromatography purification using acidic buffers (e.g., trifluoracetic acid) prior to lyophilization. Thus, care should be taken
to adjust the pH of the buffer solution upon solubilizing the
peptide. We use a benchtop pH meter equipped with microelectrode for small volume samples.
5. The concentration of peptide and protein in solution can be
measured by UV absorbance at 280 nm wavelength using a
spectrophotometer. The extinction coefficient can be calculated from the amino acid sequence (e.g., ExPASy—ProtParam) [19, 20]. A fluorophore can contribute to the
280 nm wavelength absorbance. However, the excitation and
emission wavelengths of the dansyl fluorophore are more than
10 nm away from 280 nm (λ ex ¼ 340, λ em ¼ 550); thus, the
fluorophore should not significantly affect the peptide concentration determination at the 280 nm wavelength [15]. If the
peptide lacks amino acids with chromophores, one can use the
extinction coefficient of dansyl chloride (ε ~4350 M
À1 l cm
À1 )
[21] to estimate the peptide concentration. Alternatively, one
can use a color-based protein assay (e.g., bicinchoninic acid or
Bradford assay, Thermo Scientific).
6. Dansylated peptide solutions should be kept away from light by
either covering them with aluminum foil or using tinted (light
free) microfuge tubes. Stock dansylated peptides are generally
stored in small aliquots to avoid repetitive freeze and thaw
cycles.
7. Using the PDZ protein sample immediately after purification is
highly recommended. Long-term storage of proteins at 4
C or
at À20
C can significantly affect protein stability. If storage is
required, the integrity of the PDZ sample should be tested
periodically with a known reference peptide.
8. The Hellmanex™ III (Hellma, NY) cleaning concentrate can
be used periodically to remove biological material from the
surface of cuvettes followed by thorough rinsing with ddH 2 O
and ethanol using a vacuum cuvette washer.
9. When preparing buffers for sample preparation, filtering and
degassing are highly recommended. Particulate matter and air
bubbles scatter light and disrupt the spectroscopic measurements. We filter buffers with a 0.45 μm membrane using a
vacuum filter unit attached to a dry vacuum system (Welch)
followed by continuous stirring under vacuum for 15–30 min.
146
Young Joo Sun and Ernesto J. Fuentes
protein sequence by avoiding an electrostatic dipole contribution (see [15] for additional details). In addition, the
C-terminus should be amidated to neutralize the free carboxylate group.
4. Peptides are typically purified via high-performance liquid
chromatography purification using acidic buffers (e.g., trifluoracetic acid) prior to lyophilization. Thus, care should be taken
to adjust the pH of the buffer solution upon solubilizing the
peptide. We use a benchtop pH meter equipped with microelectrode for small volume samples.
5. The concentration of peptide and protein in solution can be
measured by UV absorbance at 280 nm wavelength using a
spectrophotometer. The extinction coefficient can be calculated from the amino acid sequence (e.g., ExPASy—ProtParam) [19, 20]. A fluorophore can contribute to the
280 nm wavelength absorbance. However, the excitation and
emission wavelengths of the dansyl fluorophore are more than
10 nm away from 280 nm (λ ex ¼ 340, λ em ¼ 550); thus, the
fluorophore should not significantly affect the peptide concentration determination at the 280 nm wavelength [15]. If the
peptide lacks amino acids with chromophores, one can use the
extinction coefficient of dansyl chloride (ε ~4350 M
À1 l cm
À1 )
[21] to estimate the peptide concentration. Alternatively, one
can use a color-based protein assay (e.g., bicinchoninic acid or
Bradford assay, Thermo Scientific).
6. Dansylated peptide solutions should be kept away from light by
either covering them with aluminum foil or using tinted (light
free) microfuge tubes. Stock dansylated peptides are generally
stored in small aliquots to avoid repetitive freeze and thaw
cycles.
7. Using the PDZ protein sample immediately after purification is
highly recommended. Long-term storage of proteins at 4
C or
at À20
C can significantly affect protein stability. If storage is
required, the integrity of the PDZ sample should be tested
periodically with a known reference peptide.
8. The Hellmanex™ III (Hellma, NY) cleaning concentrate can
be used periodically to remove biological material from the
surface of cuvettes followed by thorough rinsing with ddH 2 O
and ethanol using a vacuum cuvette washer.
9. When preparing buffers for sample preparation, filtering and
degassing are highly recommended. Particulate matter and air
bubbles scatter light and disrupt the spectroscopic measurements. We filter buffers with a 0.45 μm membrane using a
vacuum filter unit attached to a dry vacuum system (Welch)
followed by continuous stirring under vacuum for 15–30 min.
146
Young Joo Sun and Ernesto J. Fuentes