Hi Florencia,

One crude way to measure this requires high speed centrifugation or ultracentrifugation facility. I would initially measure the a-Syn protein concentration in solution (day 0) and begin the aggregation. On day 6, I will centrifuge to have aggregates (fibrils) in the pellet, and measure the protein concentration in the supernatant. This will indirectly give me an idea of how much of the protein participated in the fibrillation. If the day 0 reading is considered as 100%, one can then say how much % went into aggregates from day 6 supernatant protein concentration measurement. Absorbance (A280) using UV-Vis spectrophotometer would be good for this. One day 6/7 one cannot however rule out the possibility of the availability of soluble oligomers and small aggregates in the supernatant.

Generally the fibrils are stable at room temperature. You may want to put them into 4C if you wanted to store for longer (please make sure you add 0.01- 0.02% sodium azide to your aggregation buffer to avoid microbial contamination). Additional points: 1) Sometimes the mature a-Syn fibrils may form gels in the microfuge tubes-- so don't be surprised if that happens upon prolonged incubation at room temperature! 2) Please resuspend the pellet you get from day 6 or 7 and analyze the morphology using TEM or AFM, just to make sure these are amyloid fibrils and not amorphous aggregates. 3) You may continue to do the aggregation at room temperature if you do not have 37C. However at a later point if you happen to shift your experiments into 37C, expect to see some changes in the fibrillation kinetics.

Hope this addresses most of your questions. All the best!

Anoop

Thank you Anoop Arunagiri for taking the time to answer my question so completely. I will take your advice.

You are welcome! All the best Florencia Malizia !!

Anoop