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Multiplexed Fluorescent Immunodetection Using Low Autofluorescence Immobilon®-FL Membrane
Jun Park1, Masaharu Mabuchi, Ajay Sharma
1EMD Millipore, 17 Cherry Hill Drive, Danvers, MA, 01923, USA, Jun.park@emdmillipore.com.
Methods in Molecular Biology (Clifton, N.J.)
|July 4, 2015
Summary
A new low autofluorescence membrane enhances fluorescent western immunodetection for Alzheimer's disease (AD) biomarker quantification. This method offers greater sensitivity and linear dynamic range than traditional techniques, aiding in AD research.
Area of Science:
- Biochemistry
- Neuroscience
- Molecular Biology
Background:
- Traditional chemiluminescent western blotting has limitations in signal linearity and multiplexed detection.
- High background autofluorescence in blotting membranes compromises the sensitivity of fluorescent immunodetection.
- Developing sensitive and reliable methods for biomarker quantification is crucial for understanding diseases like Alzheimer's.
Purpose of the Study:
- To introduce and evaluate a novel low autofluorescence PVDF membrane (Immobilon®-FL) for enhanced fluorescent western immunodetection.
- To assess the performance of the Immobilon®-FL membrane in quantifying Alzheimer's disease (AD) biomarkers.
- To compare the sensitivity and linear dynamic range of fluorescent immunodetection with a low autofluorescence membrane against traditional chemiluminescent methods.
Main Methods:
- Comparative analysis of autofluorescence across four commercially available blotting membranes.
- Quantitative fluorescent western immunodetection of AD biomarkers (synaptophysin, GSK3β, GAP43) using Immobilon®-FL membrane.
- Multiplexed detection and normalization to a housekeeping protein (GAPDH) for accurate biomarker quantitation in human brain samples.
Main Results:
- Immobilon®-FL membrane exhibited the lowest autofluorescence, leading to substantially increased detection sensitivity.
- Fluorescent immunodetection demonstrated a significantly wider linear dynamic range (two orders of magnitude) compared to chemiluminescent methods.
- Significantly reduced expression levels of GSK3β and GAP43 biomarkers were observed in AD brain samples compared to controls.
Conclusions:
- The combination of fluorescent immunodetection and a low autofluorescent membrane provides accurate and reliable multiplexed quantitation of AD biomarkers.
- The Immobilon®-FL membrane significantly improves detection sensitivity and linear dynamic range for western immunodetection.
- This technique is broadly applicable for protein expression analysis in various biological research contexts beyond Alzheimer's disease.
