Methodology largely will vary depending on what you're looking to do. I wouldn't recommend using DMSO as part of your solvent system if you're looking to examine the effect of various molecules on lag times, because it accelerates aggregation to a point where it becomes difficult to really see differences. Generally, in our lab, we purify our amyloid-beta via SEC in a buffer system. You could also just dilute amyloid-beta into a buffer. We then incubate samples (usually 25 uM) at 37 degrees, quiescent, with 50 mM NaCl. Aggregation can be monitored throughout the incubation, and should reach saturation within 48 hours or so. If the incubation is too fast, decrease the salt concentration or the peptide concentration. If it's too slow, do the opposite.

Many of the methods outlined by others give you excellent methods for fibril preparation, as well. The method I have detailed has the benefit of giving you plenty of time for observation of the aggregation process. It also uses a minimal amount of peptide, which, if you're using synthesized stock, greatly decreases the expense of the procedure. If you're looking for quick aggregation, though, I would try one of the methods others have mentioned.

Dear Sabrina,

I have some experience working with the amyloid beta peptide and it's aggregation. The HFIP you are talking about is used for aliquoting the Abeta into appropriate portions, where you dissolve your lyophilized Abeta in ice-cold HFIP (create ±1mg/mL), divide into portions and let the HFIP evaporate in a flume-hood. Upon usage, I usually dissolve the "film" of Abeta in a small volume of DMSO and then dilute into my incubation-concentrations. Depending on how many oligomers/fibrils you want you will use one buffer or the other (we use HCl for 24 hours at 37C for fibrils, Phosphate buffer for 24h at 4C for oligomer-enriched samples.

In our experience, the speed of aggregation can largely vary between suppliers of the peptide, so I suggest you perform some preliminary experiments in which you explore the behaviour of your own batch of Abeta.

Good luck!

This extensive article on Abeta aggregation may give you more specific details: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957213/

Thank you so much for the information. Could I contact you again if I have other question?

Dear Wesley,

do you know if dissolving the peptide in water, diluting it with PBS and incubating at 37 degrees for 24-48 h you get the aggregation? This method is right to induce the aggregation? thaks again

Abeta1-42 is monomeric in HFIP.

If you add 20% water or DMSO it will start aggregating. In 6 hours is fully aggregated. This can be easy followed by Circular Dichroism and following the alpha-helix band that is converted to beta-sheet.

Sabrina,

In general with Abeta 1-42 the hardest thing is to keep in monomeric.. it starts aggregating as soon as it comes into solution (unless you use HFIP), so the answer to your question would be: yes. Depending on the start-concentration of your Abeta and, again, the supplier, the Abeta will quickly aggregate. Mr. Orbulescu's answer may in your case be very true, that after 6h you find your Abeta aggregated... although you will likely have some monomers-dimers-trimers left in your solution. It's next to impossible to get just 1 type of aggregate unless you combine your incubation protocols with some cross-linking procedure, followed by size-exclusion chromatography (or something).

if I understood correctly, probably, the treatment with HFIP followed by evaporation is carried out to store beta amyloid in the monomeric form at -20 degree, then dissolve in DMSO and diluite into other buffer. Is it possible to diluite it then in culture medium in order to test its effect on cells in vitro?

I agree with Mr. Jongbloed,

It will be hard to get only amyloid fibrils, protofibrils will be present, dimers, trimers, tetra and hexamers. Separation of them is always an issue as the product is very expensive and the amounts are typically limited.

There is no much literature on the Abeta amyloid aggregation that one has to really narrow it down.

I did a literature search and found over 16000 articles in a single year!

@Sabrina Yes, you can. You can try to get your hands on one of our groups' publications: http://www.ncbi.nlm.nih.gov/pubmed/20544859 They performed experiments adding Abeta to cells (astrocytes) and monitoring the behaviour of the cells afterwards, either in presence or absence of amyloid associated proteins. You can vary your experiments, for instance by having Abeta aggregate WITH your interesting protein for 24 hours and adding the entire mixture to the cells, or letting your Abeta aggregate, add it to your cells and simultaneously add your interesting proteins/compounds. Each approach may cover different research questions; keep in mind that pre-incubation of proteins with amyloid beta will very likely influence it's aggregation-behaviour. It seems wise to develop an 'ideal' aggregation protocol with your Abeta1-42 and then, just before adding it to your cells, dilute it into medium ... since if you want to switch medium then at least the aggregation-state of the Abeta remains constant up to the point of adding it to your medium/cells. Different media will influence Abeta aggregation and it will be difficult to interpret your results afterwards.

The following paper may be helpful:

Biochemistry. 2003 Nov 11;42(44):12749-60.

Self-assembly of Abeta(1-42) into globular neurotoxins.

Chromy BA, Nowak RJ, Lambert MP, Viola KL, Chang L, Velasco PT, Jones BW, Fernandez SJ, Lacor PN, Horowitz P, Finch CE, Krafft GA, Klein WL.

Later, our group focuses on this topic: see

We tested different condition to obtain Oligos and Fibrils and assessed them structurally, Cerf et al 2009 http://www.ncbi.nlm.nih.gov/pubmed/19435461. Further we concentrated on the aggregation process, Sarroukh et al 2010 http://www.ncbi.nlm.nih.gov/pubmed/20853129.

For more specific details, you can e-mail me!

Good luck

Sabrina, to visualize aggregates I recommend to you use firstly of solubilization in ~5%SDS ( plus apropriate buffer) followed by BN-PAGE. The presence of charge to SDS and Coomassie G-250 will help to you to resolve aggregates without smearing. I do hope !!!

In our experience the best method to manage the beta peptide is trough the synthesis of depsipetides as shown by Beeg et al Analytical Biochemistry 411 (2011) 297–299. If its no possible to obtain depsipeptide I would not use HFIP but DMSO, to obtain relatively pure beta oligomers in our hands is sufficient to incubate in PBS 100 µM of beta 1-42 overnight at room temperature (22 °C), to obtain oligomers but also protofibers you can incubate overnight at 37 °C, a comparison between these two conditions is shown in Balducci et al PNAS 2010; 107:2295-300.

GF

I suggest the method reported in the reference below:

Fezoui Y., Hartley D.M., Harper J.D., Khurana R., Walsh D.M., Condron M.M., Selkoe D.J., Lansbury Jr P.T., Fink A.L., Teplow D.B. An improving method of preparing the amyloid β-protein for fibrillogenesis and neurotoxicity experiments. Amyloid: Int. J. Exp. Clin. Invest. 7, 166-178 (2000).

Methodology largely will vary depending on what you're looking to do. I wouldn't recommend using DMSO as part of your solvent system if you're looking to examine the effect of various molecules on lag times, because it accelerates aggregation to a point where it becomes difficult to really see differences. Generally, in our lab, we purify our amyloid-beta via SEC in a buffer system. You could also just dilute amyloid-beta into a buffer. We then incubate samples (usually 25 uM) at 37 degrees, quiescent, with 50 mM NaCl. Aggregation can be monitored throughout the incubation, and should reach saturation within 48 hours or so. If the incubation is too fast, decrease the salt concentration or the peptide concentration. If it's too slow, do the opposite.

Many of the methods outlined by others give you excellent methods for fibril preparation, as well. The method I have detailed has the benefit of giving you plenty of time for observation of the aggregation process. It also uses a minimal amount of peptide, which, if you're using synthesized stock, greatly decreases the expense of the procedure. If you're looking for quick aggregation, though, I would try one of the methods others have mentioned.

I also agree with Wesley and Matthew,

Their is not a best method, some are faster and appropriated for screening, the other re more laborious and better to have a clear picture.

I would think that a screen using Thioflavin (edit: using careful control) and FTIR spectroscopy in a first time,

then confirm results using TE microscopy and electrophoresis.

Best

Good to know, you absolutely right Ian! Thanks for correction!

@Murray is right about being cautious with ThT. We do use it extensively, but it is important to have appropriate controls, as @Nicolas said, and also to verify the aggregation via other methods. Turbidity and ANS assays both provide convenient spectroscopic methods to monitor aggregation, and can be done with equipment that is probably readily available. FTIR is also quite good, as has been pointed out, but I haven't used it extensively and am not sure how useful it would be for measuring kinetics.

FTIR or Circular Dichroism can both be used for solution work and time studies can be done therefore it can be used for slow kinetics (hours). Most of it was already studies in the late 90's.

alpha-helix band gets smaller, beta-sheet is increasing. Abeta(1-42) aggregates faster than Abetra(1-40)...about 2 hours difference until no changes are seen as a function of time;

One cannot discriminate between protofibrils and fibrils therefore no quantification can be done.

I will dig though my old data and post something if I can still locate it.

@Ian Murray, drying process does not change the aggregation at least in the concentration we used for IR (100µM) and AFM (20µM). The drying process is intensively used in EM, AFM and dot Blot and not morphological changes or immunostaining properties are observed; @ Matthew Planchard, the kinetics of aggregation can be followed by ATR-FTIR on basis of beta sheet changes (organization of strands can be followed) and on the secondary structural evolution. We have done those exp in parallel with ThT, PAGE, AFM and iummunostaining on different abeta isoforms. Additionally, CD exp are not really sensitive to learn something on beta-sheet changes when it aggregates, but is really helpful when we concentrate on helical-to-beta sheet transformamtion.

But I'm completely in agreement with you, no single technique can be used to make the kinetic study.

you may try this protocol described in a recent paper of mine.

Garai and Frieden, PNAS 2013, 110 page 3321-6

Quantitative analysis of the time course of Aβ oligomerization and subsequent growth steps using tetramethylrhodamine-labeled Aβ.

Garai K, Frieden C.

Proc Natl Acad Sci U S A. 2013 Feb 26;110(9):3321-6. doi: 10.1073/pnas.1222478110. Epub 2013 Feb 11.

http://www.ncbi.nlm.nih.gov/pubmed/23401512

hi. we recently did the experiment on this Aβ aggregation inhibition. we used a method and clearly explained in our published paper. Here I giving the DOI no .of our paper. we also concluded that monomer and oligomer forms of Aβ proteins are more toxic to the nerve cells than Aβ aggregates.

2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12732