64
1. Prepare 4 mM peptide stocks in 10 mM HCl, as described in
Subheading 3.1 (see Note 13).
2. Mix the two different peptide stock solutions (4 mM) in different ratios to a final volume of 40 μL.
3. Add 150 μL of 8 M urea to each sample, mix and incubate at
room temperature for 15 min (see Note 14).
4. Add 40 μL of 2 mM Cu
2+
in water.
5. Initiate fibril formation by adding 1.77 mL of 28.4 mM HepesKOH buffer, pH 8. Final concentrations of peptide and Cu
2+
are 80 and 40 μM, respectively (see Note 15).
6. For the blank sample, mix 40 μL of 10 mM HCl, 150 μL of
8 M urea, 40 μL of 2 mM Cu
2+
, and 1.77 mL of 28.4 mM
Hepes-KOH buffer, pH 8.
7. Set up kinetic assay as described in Subheading 3.2, steps
6–10.
4 Notes
1. Keep the solution of 1 M Hepes-KOH at 4 °C, warm up to
room temperature before use. Tris buffer coordinates to Cu
2+
and therefore should not be used in this assay. For screening at
different pH, we also used MES (pH 6) and TAPS (pH > 8).
2. Atomic absorption standard for Cu
2+
was initially used for
screening. However, this solution (1 g/L = 15.73 mM) contains 3% nitric acid and slightly reduces the final pH of samples (pH 7.9 instead of pH 8). In addition, we found that the
standard (Ricca) had high absorbance at λ below 220 nm
(even when Cu
2+
was diluted to 10 μM) and we avoided using
it as a source of Cu
2+
when measuring concentration of
Cu-peptide or when preparing samples for circular dichroism
analysis. To measure Cu
2+
concentration in CuSO 4 stock,
solution was diluted to 40 μM into 25 mM Hepes-KOH pH
8 and then Zincon in water (2 mM stock) was added to a final
concentration of 50 μM. We measured the extinction coefficient for Cu-Zincon complex to be ε 620 = 18145 M
−1
cm
−1
and dissociation constant K d = 1.7 μM. The complex of
Cu-Zincon has a maximum absorbance at 600 nm; however,
ε 620 was used to minimize contribution from unbound Zincon
(λ max = 470 nm).
3. First, dilute concentrated HCl (12 M, ACS grade) to make a
1 M HCl solution.
4. DMP solution (80 mM) in isopropanol is stable for at least 1
week at room temperature.
3.3 Mixing Peptides
with Different
Sequences
Alex Sternisha and Olga Makhlynets
1. Prepare 4 mM peptide stocks in 10 mM HCl, as described in
Subheading 3.1 (see Note 13).
2. Mix the two different peptide stock solutions (4 mM) in different ratios to a final volume of 40 μL.
3. Add 150 μL of 8 M urea to each sample, mix and incubate at
room temperature for 15 min (see Note 14).
4. Add 40 μL of 2 mM Cu
2+
in water.
5. Initiate fibril formation by adding 1.77 mL of 28.4 mM HepesKOH buffer, pH 8. Final concentrations of peptide and Cu
2+
are 80 and 40 μM, respectively (see Note 15).
6. For the blank sample, mix 40 μL of 10 mM HCl, 150 μL of
8 M urea, 40 μL of 2 mM Cu
2+
, and 1.77 mL of 28.4 mM
Hepes-KOH buffer, pH 8.
7. Set up kinetic assay as described in Subheading 3.2, steps
6–10.
4 Notes
1. Keep the solution of 1 M Hepes-KOH at 4 °C, warm up to
room temperature before use. Tris buffer coordinates to Cu
2+
and therefore should not be used in this assay. For screening at
different pH, we also used MES (pH 6) and TAPS (pH > 8).
2. Atomic absorption standard for Cu
2+
was initially used for
screening. However, this solution (1 g/L = 15.73 mM) contains 3% nitric acid and slightly reduces the final pH of samples (pH 7.9 instead of pH 8). In addition, we found that the
standard (Ricca) had high absorbance at λ below 220 nm
(even when Cu
2+
was diluted to 10 μM) and we avoided using
it as a source of Cu
2+
when measuring concentration of
Cu-peptide or when preparing samples for circular dichroism
analysis. To measure Cu
2+
concentration in CuSO 4 stock,
solution was diluted to 40 μM into 25 mM Hepes-KOH pH
8 and then Zincon in water (2 mM stock) was added to a final
concentration of 50 μM. We measured the extinction coefficient for Cu-Zincon complex to be ε 620 = 18145 M
−1
cm
−1
and dissociation constant K d = 1.7 μM. The complex of
Cu-Zincon has a maximum absorbance at 600 nm; however,
ε 620 was used to minimize contribution from unbound Zincon
(λ max = 470 nm).
3. First, dilute concentrated HCl (12 M, ACS grade) to make a
1 M HCl solution.
4. DMP solution (80 mM) in isopropanol is stable for at least 1
week at room temperature.
3.3 Mixing Peptides
with Different
Sequences
Alex Sternisha and Olga Makhlynets