I am using ThT and Congo red assays to monitor Amyloid Beta 1-42 aggregation. It worked fine before, but now I am evaluating compounds with overlapping UV absorabance to these dyes. So any suggestions to alternative assays ?
Thanks
Aggregation by design
Valda Vinson
Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al.designed a peptide based on an amyloidogenic sequence in the vascular endothelial growth factor receptor VEGFR2. The peptide induced VEGFR2 to form aggregates with features characteristic of amyloids. Amyloids were toxic only in cells that required VEGFR2 activity, suggesting that the toxicity was due to loss of function of VEGFR2, rather than to inherent toxicity of the aggregates. The peptide inhibited VEGFR2-dependent tumor growth in a mouse tumor model.
Science, this issue p. 10.1126/science.aah4949
Here is a link to a paper where we tested a range of different fluorescent dyes ThT and Congo Red against amyloids. Hope it is what you were looking for.
Article Common benzothiazole and benzoxazole fluorescent DNA interca...
Thanks a lot Daniel, this article is helpful. Is benzothiazole superior to benzoxazole in terms of amyloid beta binding ?
It doesn't really matter. It is more dependent on which wavelengths you want to use, Look at the various fluors we tried and determine which wavelengths you want to use, then you can buy them from thermo fisher.
dynamic light scattering. If you put "amyloid dynamic light scattering" into google scholar, you should see quite a few papers to get you started if you want to go this direction. This one looks attractive: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054541
fitting can be stabilized with regularization: http://scitation.aip.org/content/aip/journal/jcp/128/11/10.1063/1.2837293 (that paper is a bit technical)
Time resolved fluorescence is another approach to this if you have several spectrally overlapping fluorophores that have different lifetimes. Unfortunately ThT has several lifetimes to itself when amyloid is forming due to the multitude of binding environments.
AFM or similar techniques work if you want to take samples at time points and analyze them later.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence