Cross-linking and SDS-PAGE analysis. Aβ peptides were covalently
cross-linked using the technique of
cross-linking of unmodified proteins (PICUP), as described
31 . Briefly, 1 μL of 40 mM ammonium persulfate
(APS) and 1 μL of 2 mM Tris(2,2′- bipyridyl)dichlororuthenium(II)) were
added to 18 μL of 40 μM Aβ. The mixture was irradiated for 1s with
visible light (Dolan-Jenner Industries Fiber-Lite Model 170-D,
Boxborough, MA) and the reaction was quenched immediately with 1 μL of
1M dithiothreitol in H2O. Tricine SDS Sample Buffer (2X)
(Invitrogen, Carlsbad, CA) was then added to the solution. Five µL of
each cross-linked Aβ was analyzed by SDS-PAGE using 10–20% Tricine
gels (1.0 mm × 12 well) and subsequently silver stained following the
Silver Xpress Silver Staining Protocol (Invitrogen). Gels were
dried
for one hour using Novex DryEase Mini cellophane in Novex drying frames
and then scanned with a Canon CanoScan 9950F flatbed scanner at 300 dpi
or greater. Densitometry was performed using ImageJ 1.50d
(http://imagej.nih.gov/ij), which we used to determine the normalized
intensity of each band ; where
Ii is the intensity of band i and
is the sum of all band intensities.
Three independent experiments were performed with each peptide.
Thioflavin T fluorescence. Aβ42 and its variants were initially
dissolved in ~1 mL of 4 °C hexaflouro-2-propanol (HFIP),
sonicated 5 min to completely dissolve the Aβ, incubated for 30 min
incubation at RT, and aliquoted into a 1.5 mL low retention
microcentrifuge tube (FisherBrand). The HFIP was completely evaporated
overnight in a chemical fume hood. The following day, the tubes were
rotary evaporated for 1 h in a SpeedVac concentrator (Thermo Scientific,
Savant SPD121P), which ensured complete removal of the HFIP. The
peptides within the films then were prepared for use as described above
for peptide lyophilizates.
Immediately after sample preparation, 100 μL aliquots of each of the
peptides (40 μM Aβ40, 20 μM Aβ42) were mixed with 100 μL of 120 μM ThT
in 10 mM sodium phosphate, pH 7.4 in wells of a Thermo Scientific™ Nunc™
Microwell™ 96-Well optical-bottom plate. The plate was sealed with
SealPlate film (Excel Scientific) incubated at 37°C with shaking at 160
rpm. ThT fluorescence then was measured using a Hitachi F4500
fluorometer (Hitachi Instruments Inc., Rye, NH) with excitation and
emission filters set to 450 nm and 480 nm, respectively. Slit-widths for
excitation and emission were 5 and 10 nm, respectively. Three
independent experiments were performed for each peptide. The time at
which half-maximal ThT intensity (t1/2) was observed was
determined by calculating the difference between the final and initial
fluorescence levels, visually locating that value on a smooth fit of the
graph of the time-dependence of fluorescence intensity, and then
identifying the corresponding time.
Circular dichroism spectroscopy (CD) . Peptides were prepared as
described above for the ThT experiments. Spectra were acquired
periodically during incubation of the peptides at 37°C, without
agitation, in 0.1 cm path-length quartz cuvettes (Hellma, Forest Hills,
NY). Spectra were acquired using a Jasco Model J-810 spectropolarimeter
(Jasco corporation, Japan) over a wavelength range of ≈190–260 nm.
Parameters for measurements were: standard sensitivity, data pitch=0.2
nm, scanning speed=100 nm/min, and bandwidth=1 nm. 10 spectra were taken
per sample. Three independent experiments were performed with each
peptide.
Transmission electron microscopy (TEM). Aliquots of 8 µL volume
were removed periodically from assembly reactions being monitored by CD.
Each aliquot was applied to 400 mesh carbon-coated formvar EM grids
(Electron Microscopy Sciences, Hatfield, PA). Each grid was incubated
for 2 min at RT. The liquid was then wicked off by gently bringing the
tip of 55 mm diameter, #2 filter paper (Whatman) to the edge of the
grid. Subsequently 8 μL of 1% (w/v) uranyl acetate in MilliQ water was
applied to the grid and immediately wicked off. A JEOL 1200 EX
transmission electron microscope was used to visualize the sample
morphologies.