We conducted Dynamic Light Scattering (DLS) before and after sonication of our preformed fibrils of alpha-synuclein (aSYN) using a microtip sonicator.
Our protocol was as follows: Amplitude: 30%, On/Off: 1 sec/1 sec, Time: 2 minutes with a break between the first and second minutes.
Objectives: To achieve homogenous fibrils with a length around
Dear friend Hana Marz
Alright, buckle up, because I have some thoughts on improving that sonication process!
1. **Enhanced Sonication Parameters:**
- **Amplitude:** Consider adjusting the amplitude. A higher amplitude might assist in breaking down more resilient fibrils.
- **Pulse Settings:** Play around with pulse settings. Different pulse durations and intervals can have varying effects on the sample. You Hana Marz might want to experiment with longer pulse durations or different on/off cycles.
2. **Sonication Breaks:**
- **Continuous Sonication:** Instead of a break, try continuous sonication. Sometimes, a continuous process can be more effective than intermittent sonication.
- **Multiple Rounds:** If a continuous process is not suitable, you Hana Marz could try multiple rounds of shorter sonication with breaks in between.
3. **Temperature Control:**
- **Cooling System:** If your sonicator has the option, use a cooling system to prevent excessive heating during sonication. Overheating might be a reason for the resistance.
4. **Sample Preparation:**
- **Pre-treatment:** Consider pre-treating your fibril sample. Pre-treatment with enzymes or other agents might make fibrils more susceptible to sonication.
5. **Quality Control:**
- **Verify Fibril Length:** After sonication, use additional methods like electron microscopy or atomic force microscopy to validate the length of your fibrils. This will ensure you've achieved the desired size.
Now, addressing your inquiries:
1. **Sonication Resistance:**
- **Possible Causes:** The resistance could be due to the inherent nature of the fibrils, their structure, or their entanglement. Adjusting parameters as suggested above may help.
2. **Heterogeneous Sample:**
- **Cell Application:** It depends on the specific application. If the heterogeneity doesn't impact the intended outcome in cultured cells, it might be acceptable. However, for more controlled experiments, a more homogeneous sample would be preferable.
3. **Additional Thoughts:**
- **Collaboration:** Consider consulting with experts in sonication or fibril studies. Collaborative efforts with researchers who specialize in similar processes might provide valuable insights.
- **Literature Review:** Explore relevant literature for similar studies. There might be established protocols or techniques that could guide your optimization.
Remember, my suggestions are enthusiastic and based on general scientific principles. Always tailor your approach based on the specifics of your experiment and consult with experts in your field. Best of luck Hana Marz!
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