Related Experiment Videos
Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1
H Naiki1, K Higuchi, M Hosokawa
1Department of Senescence Biology, Chest Disease Research Institute, Kyoto University, Japan.
Abstract:
We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.
Insights
A new fluorometric method accurately quantifies amyloid fibrils in vitro. This technique uses thioflavine T dye to detect amyloidosis precursors, offering a sensitive tool for research.
Area of Science:
- Biochemistry
- Molecular Biology
Background:
- Amyloid fibrils are associated with various diseases, including murine senile and secondary amyloidosis.
- Understanding the structure and quantity of amyloid fibrils is crucial for disease research.
Purpose of the Study:
- To develop and validate a fluorometric method for quantifying amyloid fibrils in vitro.
- To assess the specificity and sensitivity of the method using different amyloid fibril precursors.
Main Methods:
- Purification of amyloid fibrils from senescence-accelerated mouse liver using sucrose density ultracentrifugation.
- Fluorometric analysis using thioflavine T dye, measuring fluorescence intensity at specific excitation and emission wavelengths.
- Assessing the effect of pH and thioflavine T concentration on fluorescence.
Main Results:
- Thioflavine T showed distinct fluorescence changes in the presence of amyloid fibrils (excitation/emission 450/482 nm) compared to its absence.
- The fluorescence intensity correlated linearly with amyloid fibril concentration (0-2.0 µg/ml) and was optimal at pH 9.0.
- Precursors of amyloid fibrils in lipoproteins showed minimal fluorescence, and fluorescence decreased upon disruption of fibril structure with guanidine-HCl.
Conclusions:
- The developed fluorometric method provides a sensitive and specific means for determining amyloid fibrils in vitro.
- This technique can differentiate between amyloid fibrils and their precursors and is robust against structural changes.