I have been setting up aggregation reaction for B2m since a long time. However, I have not been able to obtain pure (any) fibrils of B2m till now. I am briefing the condition that I have checked and its result.
Firstly about the B2m-
a. The protein has been recombinantly expressed using BL21DE3 cells.
--The protein has a methionine at the N-terminal.
b. The purity of the protein was assessed using MALDI-MS.
c. The secondary and tertiary structure has been assessed using CD Spectroscopy and Fluorescence spectroscopy, respectively.
d. The presence of di-sulfide bond in the purified protein has been assessed employing native and non-native conditions in SDS-PAGE.
Aggregation conditions for B2m-
Since my work involves experiments near-physiological conditions, I have worked to generate amyloids using copper ions. (No additives like SDS, TFE are used to keep the conditions near-physiological)
a. I have used (as per the reported literature) 100 uM of B2m, 200 uM of CuSO4, 200 mM of Potassium acetate, 25 mM MOPS, 500 mM Urea and incubated the reaction at 37 deg C without agitation. However, I have not been able to get any amyloids. There is no increase in ThT intensity with days. I have checked it at 10th day and 20th day of the experiment using TEM. I have attached few TEM pictures for consideration.
b. I am now working with acid conditions to generate amyloids of B2m and use their seeds at physiological pH.
1. Can anyone help me to understand where things are going wrong?
2. If there is any particular conditions that might be set?
3. I have observed increased ThT intensity once in my initial try to generate fibrils using the same conditions as explained above, later confirmed using AFM (pic attached). However, it seemed a heterogeneous population of aggregates then. But amyloids did form. However, I have not been able to replicate the experiment with then obtained results.
4. The N-terminal being involved in copper binding is what I feel might be an issue. After setting the aggregation reaction at above conditions now I always get precipitate in the control (No copper, but 10 mM EDTA) and the reaction tube within 10 minutes (which was not the case during my initial try).
*I have tried replicating the initial conditions for generating amyloids. I purify protein using PBS pH 7.4, which later, I buffer exchange into 25 mM MOPS pH 7.4 using SEC before setting the aggregation reactions using the above condition.
200 µM CuSO4 isn't exactly physiological, so I'd not worry too much about other unphysiological additions. Amyloid fibre formation has a lag-time as small clusters of molecule break down faster than they form (and a good thing that is, too, otherwise we'd all be dead). Only once nuclei of a certain size have been formed, do you get measurable aggregation. So, may be, you just need to push the molecules a little harder or wait a little longer?
Engelbert Buxbaum
Hi, Thanks for the reply.
Yes, the concentration of CuSo4 that has been used in prior optimization of setting aggregation reaction (as per already available literature) isn't physiological and the concentration of B2m being used isn't as well. It is the ratio 1:2 (B2m:Cu(2+)) which has been observed as important.
Can with time the amorphous-like aggregates turn into mature amyloid fibrils? I have observed the aggregation reaction till 20th day (which is more than 2 weeks, as per the provided information in the literature). However, I will look into it and wait for some more days.
"... push the molecules a little harder..." I had tried PBS and MOPS buffer, at different concentration of proteins (10, 20, 30 ,....100 uM B2m). In all the set reactions 'good amount of precipitation' was common, clearly visible within 10 minutes of setting the reaction. And ThT binding assay performed for 10 days of incubation showed no increase in intensity.
The very first successful amyloid were obtained in PBS, which I continued for some experiments. However, after no reproducibility and upon learning phosphate being a chelator for divalent ions. I switched to MOPS.
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