66
Different plate readers could have different correction
coefficients even when the same plate and the same volume
are used.
8. 50 mM solution of CuSO 4 in water is stable for at least a
year; the 1 mM stock was prepared immediately before kinetic
experiment.
9. It is important to mix the peptide and Cu
2+
at a low pH, before
fibrils are formed. First, mix peptide (pH 2 stock) and Cu
2+
solution in water, then add Hepes-KOH buffer, pH 8. Using 28.4 mM
Hepes-KOH will give a 25 mM final concentration of buffer in
the sample. The change in pH from 2 to 8, and the presence of
Cu
2+
, triggers fibril formation. Always make Cu- peptide samples
in pH 8 buffer immediately before the experiment; we observed
reduced catalytic activity over time due to fibrils binding to plasticware. Some peptides need prolonged incubation time to form
fibrils. Optimal incubation time should be established for each
peptide family. The easiest approach would be to screen for
peptide activity using freshly prepared Cu-peptide samples, then
incubate leftover sample for 24 h and repeat the screen.
10. Avoid formation of bubbles as they will interfere with absorbance measurements.
11. Final concentrations of the reagents in the well at the onset of
the reaction are: 10 μM Cu
2+
, 20 μM peptide, 500 μM DMP,
2.2% isopropanol, 18.8 mM Hepes-KOH, 0.26 mM dioxygen
(the solubility of dioxygen in air-saturated water at 25 °C and
760 Torr [23]). With DMP substrate we observed that after a
minute, the kinetic trace at 476 nm starts to curve; therefore
losing a few data points in the beginning of the reaction can
have a significant effect on the calculated rate. If peptides are
very active, it is recommended to reduce the concentration of
DMP to 200 μM (final concentration) or lower.
12. Conditions described above use stoichiometric amount of Cu
2+
needed to saturate all binding sites in fibrils. Dividing the rate
calculated for Cu-peptide sample by the rate of the blank sample will show rate enhancement of the reaction in the presence
of peptide.
13. Some peptides might not be soluble enough in 10 mM HCl to
make 4 mM solution. In this case, peptides can be weighed out
and then dissolved in 8 M urea to make 4 mM solution without
measuring absorbance at 214 nm (urea absorbs at 214 nm).
14. Urea breaks fibrils and this step ensures that the two peptides
mix properly. Without urea treatment, peptides in 10 mM HCl
might exist as protofibrils and would not mix well enough to
result in uniform fibril composition.
15. To initiate fibril formation, urea needs to be diluted at least
10-fold.
Alex Sternisha and Olga Makhlynets
Different plate readers could have different correction
coefficients even when the same plate and the same volume
are used.
8. 50 mM solution of CuSO 4 in water is stable for at least a
year; the 1 mM stock was prepared immediately before kinetic
experiment.
9. It is important to mix the peptide and Cu
2+
at a low pH, before
fibrils are formed. First, mix peptide (pH 2 stock) and Cu
2+
solution in water, then add Hepes-KOH buffer, pH 8. Using 28.4 mM
Hepes-KOH will give a 25 mM final concentration of buffer in
the sample. The change in pH from 2 to 8, and the presence of
Cu
2+
, triggers fibril formation. Always make Cu- peptide samples
in pH 8 buffer immediately before the experiment; we observed
reduced catalytic activity over time due to fibrils binding to plasticware. Some peptides need prolonged incubation time to form
fibrils. Optimal incubation time should be established for each
peptide family. The easiest approach would be to screen for
peptide activity using freshly prepared Cu-peptide samples, then
incubate leftover sample for 24 h and repeat the screen.
10. Avoid formation of bubbles as they will interfere with absorbance measurements.
11. Final concentrations of the reagents in the well at the onset of
the reaction are: 10 μM Cu
2+
, 20 μM peptide, 500 μM DMP,
2.2% isopropanol, 18.8 mM Hepes-KOH, 0.26 mM dioxygen
(the solubility of dioxygen in air-saturated water at 25 °C and
760 Torr [23]). With DMP substrate we observed that after a
minute, the kinetic trace at 476 nm starts to curve; therefore
losing a few data points in the beginning of the reaction can
have a significant effect on the calculated rate. If peptides are
very active, it is recommended to reduce the concentration of
DMP to 200 μM (final concentration) or lower.
12. Conditions described above use stoichiometric amount of Cu
2+
needed to saturate all binding sites in fibrils. Dividing the rate
calculated for Cu-peptide sample by the rate of the blank sample will show rate enhancement of the reaction in the presence
of peptide.
13. Some peptides might not be soluble enough in 10 mM HCl to
make 4 mM solution. In this case, peptides can be weighed out
and then dissolved in 8 M urea to make 4 mM solution without
measuring absorbance at 214 nm (urea absorbs at 214 nm).
14. Urea breaks fibrils and this step ensures that the two peptides
mix properly. Without urea treatment, peptides in 10 mM HCl
might exist as protofibrils and would not mix well enough to
result in uniform fibril composition.
15. To initiate fibril formation, urea needs to be diluted at least
10-fold.
Alex Sternisha and Olga Makhlynets