Multiplexed Detection

Cell signaling pathways are often regulated by protein phosphorylation cascades. A protein such as a receptor tyrosine kinase becomes phosphorylated following ligand binding setting off multiple signaling pathways. Highly sensitive two-color NIR fluorescence detection enables the simultaneous detection of the total protein present as well as the amount of protein phosphorylation that has occurred without the need for stripping and reprobing – something that cannot be done with chemiluminescence. This approach to multiplexed detection has been implemented in the Odyssey® and Aerius™ infrared imaging systems.  These instruments both use two separate NIR lasers and detectors to image labeled antibodies at 710 and 805 nm, simultaneously.

A.	Overlaid Images Total EGFR Phosphorylated EGFR (700 nm)
B.	800 nm Image Total EGFR
C.	700 nm Image Phosphorylated EGFR
Anti-EGFR and anti-phospho-EGFR antibody specificity in A431 cells. Two-fold serial dilutions of unstimulated and EGF-stimulated A431 cell lysates are shown in two-color images collected with an Odyssey Infrared Imaging System. Single color images (B and C) can be overlaid (A) to show both total protein and phosphorylated protein (yellow indicates overlapping red and green signals). The mobility shift caused by phosphorylation is visible (A) as indicated by the red bands above the yellow bands.

This simultaneous ratiometric approach to multiplexed detection greatly increases the accuracy of quantitative immunoblotting. One detection channel can be used to detect a protein of interest and a second channel to normalize for sample loading; for example, detecting the amount of phosphorylated protein in one channel and a housekeeping protein in the second.  The ratio of the signal from an unknown protein to a housekeeping protein can be used to accurately normalize the signal intensities and correct for loading and sampling errors.

Two-channel NIR fluorescent detection provides the unique capability to accurately quantify proteins over a large dynamic range.  Very low amounts of proteins have been accurately quantified using this ratiometric approach. This accuracy is difficult to achieve with other detection methods. While chemiluminescent detection has good sensitivity, its limited dynamic range, and lack of a second detection channel for ratiometric analysis make it very difficult to accurately quantify low amounts of proteins.