I am carrying out ThT - amyloid fibril binding assay at different pH (buffer system) with same beta amyloid concentration, surprisingly the fluorescence intensity is different in each case.
I thought it might be due to the different oligomeric species at different pH so I have tried out seeding experiment but still there is no change in fluorescence intensity.
What would be the probable reason for such change in fluorescence intensity and what would be best pH (buffer system) to carry out this experiment?
Hi Arun,
Your results may not be surprising! The change in fluorescence intensities with at different pH values could be due to the disintegration of fibrils at those pH. I am not sure what pH range you are studying but the fibrils are likely to precipitate out as soon as the isoelectric point is reached.
The ThT assay works well at alkaline pH than at high acid conditions. Hence you may find that buffers for this assay are mostly in the near neutral range. There are numerous papers on the methods and I am sure you may be following one or more of these. If you need help just let me know.
We have worked with Beta Lactoglobulin fibrils and for the ThT assay the buffer was pH 7 phosphate buffer (10 mM Phosphate+150 mM sodium chloride). In our case the fibrils were made at acidic pH (pH 2) but the ThT assay was at pH 7. The fibrils from this protein are fairly stable over the pH range, barring a small patch between pH 4.5 to 5.5.
Also if you have an interest in amyloid fibrils and related aspects, and if you haven't already seen it, you can follow the discussion on a project on this website:
https://www.researchgate.net/project/Amyloid_open_protocols/
There are some useful details about the protocols on this forum which may be of help to you. If you need any more information specifically do feel free to write back.
Good luck,
AD
It is difficult to compare fluorescent intensities across almost any environmental variable since the quantum yield of the bound dye, the binding constant, the stabilty of the dye, and population of oligomeric species can all change. The effect of the quantum yield can minimized by using fluorescent polarization measurements, the other effects are hard to minimize. Generally it is more accurate to use shifts in the kinetics rather than the actual intensities.
K114 probably will have less pH dependence than thioflavin T although I am not aware that it has been tested
http://onlinelibrary.wiley.com/doi/10.1046/j.1471-4159.2003.01949.x/abstract
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