62
of this solution should be confirmed using ICP or by measuring
absorbance of Cu-Zincon complex (see Note 2).
3. 10 mM HCl (see Note 3).
4. 80 mM 2,6-dimethoxyphenol (DMP): weigh out 20–30 mg of
DMP solid is a microcentrifuge tube, add 1 mL of isopropanol,
and invert the tube to dissolve the solid. Further dilute the
resulting solution using isopropanol to a final concentration of
80 mM (see Note 4).
5. 8 M urea solution in water: weigh out 2.4 g of urea, then add
water to 5 mL.
6. Isopropanol.
7. 96-well plates.
8. Multichannel pipette.
9. UV-Vis spectrophotometer.
10. Plate reader.
3 Methods
Peptides were synthesized by manual Fmoc solid-phase synthesis at
elevated temperature using Rink Amide or Wang resin and Fmocprotected amino acids using a previously reported protocol [20].
The crude peptides were precipitated and washed with cold methyltert- butyl ether, and purified using reverse phase High Performance
Liquid Chromatography. The identities of the peptides were confirmed using MALDI-TOF mass spectrometer and the purity was
evaluated on analytical HPLC instrument. Pure (>90%) lyophilized
peptides were stored at −20 °C (see Note 5).
1. Calculate the extinction coefficient of peptide at 214 nm
(see Note 6).
2. Prepare a 1 mM stock solution of peptide in 10 mM HCl.
Using the Beer-Lambert law, calculate the concentration of the
peptide that will give an absorbance at 214 nm between 0.2
and 1. Dissolve a small amount of solid peptide in 1 mL of
10 mM HCl, dilute the stock of peptide using 10 mM HCl
(measure at least 10 μL of peptide, perform serial dilutions for
peptides with high ε 214 ), and measure the absorbance. If the
concentration is above 1 mM, dilute the stock using 10 mM
HCl. If the concentration is less than 1 mM, add more solid
peptide. Some peptides do not dissolve completely. If this is the
case, centrifuge the solution at 10000 × g for 5 min, carefully
remove the supernatant, and then measure concentration. Once
the stock is at 1 mM, perform dilutions and retake absorbance
measurement at least one more time to ensure reproducibility.
3.1 Preparation
of Peptide Stock
Solution at pH 2
Alex Sternisha and Olga Makhlynets
of this solution should be confirmed using ICP or by measuring
absorbance of Cu-Zincon complex (see Note 2).
3. 10 mM HCl (see Note 3).
4. 80 mM 2,6-dimethoxyphenol (DMP): weigh out 20–30 mg of
DMP solid is a microcentrifuge tube, add 1 mL of isopropanol,
and invert the tube to dissolve the solid. Further dilute the
resulting solution using isopropanol to a final concentration of
80 mM (see Note 4).
5. 8 M urea solution in water: weigh out 2.4 g of urea, then add
water to 5 mL.
6. Isopropanol.
7. 96-well plates.
8. Multichannel pipette.
9. UV-Vis spectrophotometer.
10. Plate reader.
3 Methods
Peptides were synthesized by manual Fmoc solid-phase synthesis at
elevated temperature using Rink Amide or Wang resin and Fmocprotected amino acids using a previously reported protocol [20].
The crude peptides were precipitated and washed with cold methyltert- butyl ether, and purified using reverse phase High Performance
Liquid Chromatography. The identities of the peptides were confirmed using MALDI-TOF mass spectrometer and the purity was
evaluated on analytical HPLC instrument. Pure (>90%) lyophilized
peptides were stored at −20 °C (see Note 5).
1. Calculate the extinction coefficient of peptide at 214 nm
(see Note 6).
2. Prepare a 1 mM stock solution of peptide in 10 mM HCl.
Using the Beer-Lambert law, calculate the concentration of the
peptide that will give an absorbance at 214 nm between 0.2
and 1. Dissolve a small amount of solid peptide in 1 mL of
10 mM HCl, dilute the stock of peptide using 10 mM HCl
(measure at least 10 μL of peptide, perform serial dilutions for
peptides with high ε 214 ), and measure the absorbance. If the
concentration is above 1 mM, dilute the stock using 10 mM
HCl. If the concentration is less than 1 mM, add more solid
peptide. Some peptides do not dissolve completely. If this is the
case, centrifuge the solution at 10000 × g for 5 min, carefully
remove the supernatant, and then measure concentration. Once
the stock is at 1 mM, perform dilutions and retake absorbance
measurement at least one more time to ensure reproducibility.
3.1 Preparation
of Peptide Stock
Solution at pH 2
Alex Sternisha and Olga Makhlynets