( filter for Cy5-conjugated antibodies an...)Multiplex protein detection with the ECL Plex fluorescent Western blotting system using the Ettan DIGE Imager

Methods—multiplex protein detection
Model system
A protein mixture of human apotransferrin and bovine cardiac muscle actin was loaded onto Novex 12% Tris-glycine gels in four-fold dilutions from 5 ng to 1.2 pg (transferrin) and in two-fold dilutions from 150 ng to 2.34 ng (actin). The electrophoresis, protein transfer, blocking, and washing steps were identical to the single protein detection protocol. Both the Hybond ECL and Hybond-LFP membranes were used.

After the blocking step, the blots were incubated in a mixture of rabbit anti–human transferrin and mouse anti–bovine actin primary antibodies (diluted 1:750 in PBST) for 1.5 h at room temperature. After washing in PBST, the blots were incubated with a mixture of the secondary antibodies, ECL Plex goat-α-rabbit IgG-Cy5 and ECL Plex goat-α-mouse IgG-Cy3 (both diluted 1:2500 in PBST), on a shaker for 1 h at room temperature and protected from light.

The membranes were then scanned on the Ettan DIGE Imager and the Typhoon scanner in single scans applying both Cy3 and Cy5 wavelengths and filters. Limit of detection and cross-reactivity between the two CyDye-antibody conjugates were analyzed for all alternatives.

Application
TGF-β is a potent growth factor stimulating a number of cellular responses including growth inhibition, cell differentiation, and apoptosis. In this application, human T293 kidney epithelial cells were activated with TGF-β and harvested at different time points. Lysates of these cells wer
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