Multiplexed Fluorescent Immunodetection Using Low Autofluorescence Immobilon®-FL Membrane.

Abstract

By enabling greater signal linearity and multiplexed detection, fluorescent western immunodetection overcomes many of the inherent technical limitations associated with the traditional chemiluminescent detection method. However, the sensitivity of fluorescence detection can be severely compromised by high background autofluorescence of various blotting membranes. Here, we describe a low autofluorescence PVDF membrane (Immobilon(®)-FL membrane) optimized for fluorescent immunodetection, and we report its use in the quantitative fluorescent western immunodetection of biomarkers associated with Alzheimer's disease (AD). First, membrane autofluorescence of four different commercially available blotting membranes was compared. Immobilon(®)-FL membrane exhibited the lowest autofluorescence with substantially increased detection sensitivity. We also show that the fluorescent immunodetection exhibited greatly increased linear dynamic range (two orders of magnitude, log scale) compared to the traditional chemiluminescent methods (less than one order of magnitude). Immobilon(®)-FL membrane was then used to quantify the expression levels of previously reported biomarkers associated with AD (synaptophysin, GSK3β, and GAP43). Total protein extracts from age-matched brain samples of three AD patients and three normal controls were used. Biomarker expression levels were normalized to that of a housekeeping protein (GAPDH) using multiplexed detection, conserving difficult-to-obtain biological tissue samples and minimizing experimental variation. We found that the expression of GSK3ß and GAP43 biomarkers were significantly reduced in AD brain samples compared to age-matched normal samples. In summary, combining fluorescent immunodetection with a low autofluorescent blotting membrane yields accurate and reliable multiplexed quantitation of AD biomarkers. While this report focuses on the quantitation of AD biomarkers, the described technique is applicable to comparing protein expression in other biological contexts as well.