Quantitative, Two-Color Western Blot Detection With Infrared Fluorescence

Accurate and reproducible quantifi cation of proteins on Western blots across a wide linear range is desirable, but diffi cult to achieve. Chemiluminescent detection is subject to enzyme/substrate kinetics that may affect performance, and use of antibodies labeled with visible fl uorophores has not provided the necessary sensitivity for practical use. In this study, we investigated the use of antibodies directly labeled with near-infrared (IR) dyes for Western blot detection. Sensitive detection was achieved, presumably due to very low autofl uorescence of membranes in the IR range compared to the visible light range, with 200-fold greater sensitivity than previous studies with visible fl uorophores and greater sensitivity than chemiluminescence. We compared the linear range of IR detection to chemiluminescence in a dot blot assay, and found that IR fl uorescence provided a 16-250 fold wider quantifi able linear range with increased reproducibility. Finally, we have demonstrated the use of two different IR fl uorophores for simultaneous, multiplexed detection of two proteins at endogenous levels in whole cell lysates.