Has anyone successfully seen Abeta fibrils in a Tris buffer within 72hrs? I'm currently doing ThT assays with 20uM Abeta40, Tris buffer, 20uM ThT and @ 37C for 72hrs. I'm not seeing anything and I have no idea why. Everyone seems successful with PBS as a buffer and say that Tris buffers work equally well too, but I haven't found this so called success with the Tris buffer. Any help would be great I am seriously confused by my results thus far.
FYI my Abeta disaggeration protocol is HFIP treatment to produce film and reconstitute with 60mM NaOH (I checked this is okay pH-wish in my buffer)
Are the sample being shaken? Without shaking, it takes around 10 days for fibers to form (see fig 4a http://www.sciencedirect.com/science/article/pii/S0022283601949708 and fig 4 https://www.researchgate.net/publication/235748979_Resolution_of_Oligomeric_Species_during_the_Aggregation_of_A1-40_Using_19F_NMR?ev=prf_pub
Tris and phosphate should give equivalent kinetics for Abeta (appears not to be true for some other proteins like IAPP)
60 mM NaOH is excessive. You only need to bring the pH to 10 (1mM NaOH maybe 10 mM to be safe). The extra salt concentration can have an effect if not adjusted for
http://www.ncbi.nlm.nih.gov/pubmed/15683229
Article Resolution of Oligomeric Species during the Aggregation of A...
Okay thanks. I'm gonna carry these incubations out longer then and see if I find my fibrils after 7-10 days instead of the first 48
So I was able to find fibrils after 10-13 days from the start of incubation. The issue I feel was the lack of agitation over the time period. The fibrils I found were very long (>or= to 5um). In addition my ionic strength was a little high compared to other experiments in the literature, which can prolong the time for fibril formation. Given that I will be doing similar in solution on the AFM I think the best course is to add agitation to my protocol. However I will not be able to add agitate on the AFM. I wonder if this will be a large issue when comparing ThT and AFM data in may paper. Curious what others thoughts are.
Hi, you can enhance fibril formation of AB peptide using a micro stir bar and a 1 cm polystyren cuvettes. Has been reported that fibril formation occurs faster when you use plastic cuvettes. Of course, 37C, in presence of ThT.
Never heard of anyone using a stir bar and cuvettes. If I just wanted to make fibrils there are easier ways to get fibrils that is well established. I don't just want fibrils I'm looking for morphological changes of the aggregates under different conditions.
Stiring it with a stir bar breaks up the fibrils that form in aanner I don't want
The fibers formed from shaking aremorphologically different http://pubs.acs.org/doi/full/10.1021/bi05195 . Compare the solid-state NMR structures with shaking have two fold symmetry (pdb 2LMO and 2LMN) while those performed without shaking have 3 fold (pdb 2LMP and 2LMQ). The shaking fragments the fibers and enhances secondary nucleation resulting in shorter fibers and also mixes air into the solution, creating an additional hydrophobic interface
http://pubs.acs.org/doi/full/10.1021/ja102896h.
More specifically, this can be an issue in publication. I have this question from a reviewer on a paper I am trying to publish right now. The only drawback to not shaking is the greater spread in aggregation time and lower reproducibility. If you don't need a quantitative comparison of kinetics in two conditions that probably won't matter. If you do, increase the number of replicates (at least 6).
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