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Qualitative and quantitative comparison of brain proteins in Alzheimer's disease
L Buee1, A Laine, A Delacourte
1Unité No. 16 INSERM, Laboratoire de Neurosciences, Lille, France.
Insights
Immunoblotting accurately quantifies insoluble Alzheimer's disease proteins like GFAP and Tau. However, its reliability for soluble proteins like alpha 1-antichymotrypsin is uncertain when compared to other methods.
Area of Science:
- Neuroscience
- Biochemistry
- Pathology
Background:
- Alzheimer's disease (AD) pathology involves insoluble proteins in brain extracts.
- Immunoblotting is commonly used for analyzing denatured and reduced proteins in AD research.
- Accurate protein quantification is crucial for understanding AD etiology.
Purpose of the Study:
- To assess the quantitative accuracy of immunoblotting for Alzheimer's disease proteins.
- To compare immunoblotting results with electroimmunoassay for soluble proteins.
- To investigate the levels of Glial fibrillary acidic protein (GFAP), Tau proteins, and alpha 1-antichymotrypsin in AD brains.
Main Methods:
- Analysis of brain homogenates from control and AD patients using immunoblotting.
- Application of Laemmli sample buffer for protein denaturation and reduction.
- Densitometric analysis of protein bands to assess linearity and quantity.
- Comparison of immunoblotting results with electroimmunoassay for specific proteins.
Main Results:
- Demonstrated linearity of densitometric measures for immunoblotting.
- Established an 8 to 16-fold increase in GFAP in Alzheimer's brains.
- Identified two pathological Tau variants (Tau 64 and 69) proportional to neurofibrillary degeneration.
- Observed discrepancies between immunoblotting and electroimmunoassay for alpha 1-antichymotrypsin.
Conclusions:
- Immunoblotting is reliable for quantifying insoluble proteins in Alzheimer's disease brain extracts.
- Quantitation of soluble proteins by immunoblotting may be uncertain when compared to other methods.
- The study confirms increased GFAP and pathological Tau variants in Alzheimer's disease brains.
Abstract:
In human brain extracts, most proteins of pathological interest in Alzheimer's disease are insoluble and their analysis is often performed on denatured and reduced samples by immunoblotting after electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulfate. Because we needed to accurately compare the concentration of several proteins in brain extracts to investigate the etiology of the disease, the quantitative aspect of immunoblotting was assessed and the results compared for a soluble component with those obtained by electroimmunoassay. Glial fibrillary acidic protein (GFAP) and Tau proteins were analysed by immunoblotting in brain homogenates treated with the Laemmli sample buffer from 10 control and 25 Alzheimer's disease brains. The linearity of densitometric measures of dilutions for one given sample was demonstrated. A 8 to 16-fold GFAP increase in Alzheimer brain was established. With regard to Tau proteins it was possible to show the presence of two pathological Tau variants (Tau 64 and 69) in all the Alzheimer brain homogenates, furthermore, the amount of Tau 64 and 69 was proportional to the presence of neurofibrillary degeneration. As far as alpha 1-antichymotrypsin is concerned, we showed, in a second set of brain samples (14 control and 12 Alzheimer brains), discrepancies between the results obtained by immunoblotting and by electroimmunoassay while for a given sample linearity of immunoblotting measures of dilutions of this sample was demonstrated. Quantitation by immunoblotting of such components which can be quantified using other procedures is uncertain whereas the interest of immunoblotting is undoubted for the insoluble proteins in the brain extracts.