I've always wondered about the relevance of this. I mean, 800 rpm would have to be a pretty fast & continuous hula hooper. When we were working on synuclein, some 5-7 years ago, we used to use 250 rpm at 37C and had huge problems forming fibrils. Without agitation it would take a month to form fibrils and if you follow the reaction by 15N-NMR you can see synuclein usually starts to have all kinds of degradation products over that timescale (of course this would depend on purification scheme). It's pretty much why we worked on non-fibrillar states of synuclein and switched to other amyloids. We found a key issue in synuclein fibrillization is a boiling step in the purification where you heat the cell lysate to get rid of contaminating proteins. We did countless mass spec experiments (unpublished) to prove to ourselves nothing was happening to the covalent structure (like deamidation) but it made a huge difference in the speed of fibrillization whether the samples were boiled or not. Presumably heat was non-covalently disrupting some fibrillatization nucleus, and since these are nucleation reactions, it doesn't take much. I think others may have published on this more recently.

Hi, thanks for your answer. Yes, many have published recently and i am working on purified synuclein so purification part wont be a problem. Only issue is the amount of sample in the incubation tube. If its 30-50 microlitre and if that agitation speed (600-800rpm) is enough for the liquid in the tube to agitate to form oligomers?  

There was nice paper a few years ago looking how different variables affect reproducibility in plate assays (including shaking speed & volume)

http://www.ncbi.nlm.nih.gov/pubmed/20149780

Thanks. Its a good paper

Hello,

I am working in synuclein aggregation and fibrillogenesis for many years!

the sensitive steps in the aggregation are:

1. concentration

2. agitation

Volume is not a problem, but you need to have a protein concentration higher then 10uM and the agitation should be not so fast otherwise you will have troubles to get fibrils. I suggest you a middle agitation 

Best

Hi Simona

Thanks for your suggestion. I use 600 rpm and the volume in my tubes were 30 microlitre to 100 microlitre and the sample concentration is more than 25 uM. After incubation for 14 days, when i did CD Spec, it was still monomers. I am thinking of pooling the samples (with same concentration) and do rotating incubation. I am also worried the samples might have sticked to the walls of eppendorf tube? Does the incubating container play a significant role?

Actually it does - you should get low-retention Eppendorfs!

My experiences were similar. You could see fibrils by EM but a lot of the sample was still monomers. Again, I think it has to do with the ailing purification step. Unboiled samples seemed to fibrillize faster.

Hi Andrei

I am trying to make fibrils from purified recombinant protein that is commercially available so i am not expression the protein and purifying. 

That one worked better in my experience.

It also helps to add a teflon beed when you're shaking.

Thanks Andrei

Hi Richard,

I was just inquiring if you had any success in forming oligomers from the commercially bought synuclein? I was just planning to buy some to try and form oligomers. Thank you.

Hi,

what is the buffer that you are using to dissolve the commercial recombinant protein?Could be important and I can suggest you an appropriate buffer for the synuclein aggregation! is the protein tagged? it is very important to buy protein without any tag otherwise you can't get fibrils

Hi Simona, i use pbs to dissolve protein and also the protein is not tagged.

Hi Megha, yes, i have success in making oligomers from commercially available synuclein. All d best . 

Hi Richard, Thank your for the response. Could you please tell me the minimum concentration and volume of protein you use to form oligomers in PBS. Most papers i read mention a starting concentration of 3- 5mg/ml and agitation. Is this high a  starting concentration required to form oligomers? Would it be possible for you to give me the protocol with which you have had success.

Hi Richard, were you successful in making the alpha synuclein fibrils from the commercially bought protein. And how exactly did you bring about the agitation in the solution. I use the similar concentration and volume as you did, in Tris-HCl buffer. But  I have had no success in achieving the fibrillation.

We have been successful in making active alpha synuclein fibrils (aggregates) and monomers. They have been tested and found to induce Lewy body pathology in primary rat hippocampal neurons.

Here's a list of the 4 alpha synuclein proteins we offer:

Active Human Alpha Synuclein Aggregates (Catalog No. SPR-322)

http://www.stressmarq.com/products/protein/alpha-synuclein-protein-spr-322/

Active Human Alpha Synuclein Monomers (Catalog No. SPR-321)

http://www.stressmarq.com/products/protein/alpha-synuclein-protein-spr-321/

Control Human Alpha Synuclein Aggregates (Catalog No. SPR-317)

http://www.stressmarq.com/products/protein/alpha-synuclein-protein-spr-317/

Control Human Alpha Synuclein Monomers (Catalog No. SPR-316)

http://www.stressmarq.com/products/protein/alpha-synuclein-protein-spr-316/

Dear all,

I am also trying to making aSyn aggregates and I am wondering what is in your opinion the best buffer of choice? Is PBS sufficient or is a buffer containing KCl/NaCl or Tris better?

Article Best Practices for Generating and Using Alpha-Synuclein Pre-...

Have a nice day, for any doubt I'm on twitter. @gasparottoJG

300 uM aSynuclein with 150 mM NaCl in 20 mM Tris buffer pH 8.0 resulted in more than 90% fibrils in 6 days. It requires constant agitation of 600 rpm at 37 degree Celsius. So far I know, 100 uM aSyn concentration should also be fine.