eBook: Advancing Neuroscience Research - 20

Effects of Sonication on
alpha-synuclein PFFs
* Size: Sonication reduces the size of
PFFs into smaller segments
7. Thaw the rest of the frozen stored fibrils at
room temperature.
* Solubility: Sonication makes
PFFs soluble
Sonication settings & protocols
Sonication protocols differ based on the type and
model of sonicator used. Regardless of the sonication
method and type used, the parameters should
always be validated with transmission electron microscopy
(TEM) to confirm average fibril length of
~50 nm (for alpha-synuclein PFFs).
The following steps are suggested as a starting
point to determine the optimal sonication settings
prior to using pre-formed fibrils (PFFs).
1. Store fibrils at -80°C as soon as they
are received.
2. Plan out different sonication parameters
(i.e., fibril volume).
3. Thaw the amount of fibrils needed.
4. Aliquot the thawed fibrils into multiple
sonication tubes and sonicate them at the
different times and settings accordingly with
the settings made in step 2.
5. Observe fibrils through TEM and determine
which sample has fibrils with a ~50 nm size
distribution.
6. Record the optimal sonication settings of the
sample determined in step 5.
8. Sonicate at the optimal settings as
recorded in step 6. Optimally, the samples
are sonicated shortly before using them.
Alternatively, aliquot the sonicated samples
in the volumes that are to be used in
experiments and store them at -80°C.
If using a probe sonicator, a starting point could
be similar to the protocol used by Volpicelli-Daley/Michael
J. Fox Foundation,1
which is sixty 0.5
second pulses with a 5 second pause every 10
pulses (20% power).*
Bath sonicators are generally preferred when dealing
with low volumes of samples. Since each sonicator
and each experiment is different, researchers
will need to determine the best settings. The settings
below have been reported by some researchers
as a starting point for sonicating alpha-synuclein
PFFs using common bath sonicators to achieve a
fibril length of ~50nm.
However, *parameters should always be validated
with transmission electron microscopy (TEM) to
achieve desired fibril length according to individual experimental
parameters and conditions.
* Bransonic series, model M1800 Bath
Sonicator: Sonicate approximately 10 μL
of sample (5 mg/mL) in a 0.5mL centrifuge
tube, continuously for 1h.*
* QSonica Bath Sonicator: Sonicate at 80%
amplitude (30 secs OFF, 30 secs ON for 30
cycles) at 10°C.*
* Diagenode Bioruptor® Pico Bath
Sonicator: Sonicate 30s on, 30s off, 5 cycles
high frequency using 1.5 ml Bioruptor®
Pico Microtubes with Caps (C30010016) in a
minimum volume of 100 uL at 10°C.*
20
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