Increased Information from Multiplex Fluorescent Westerns
Analysis of multiple proteins using chemiluminescence typically involves stripping and sequentially reprobing blots for each additional target of interest. This method is time-consuming, and uneven stripping of the blot can cause loss of quantitative information. A recent alternative to chemiluminescence is the use of fluorescently labeled secondary antibodies.
Fluorescent detection makes it possible to assay multiple proteins simultaneously with fluorescently labeled secondary antibodies that have nonoverlapping excitation and emission spectra (Figure 2). Figure 3 depicts a two-color Western blot imaged using the FluorChem Q, in which two proteins were detected using SpectraPlex™ secondary antibodies.
Simultaneous analysis of multiple proteins on a single blot allows a loading control or internal standard to be assayed alongside a protein of interest for each sample, improving the quantitative accuracy of the data obtained from the blot.
Figure 4 depicts a three-color Western blot, in which the detected antibodies were labeled with CyDyes™ (GE Healthcare) and fluorescein (Vector Laboratories). The amount of transferrin (red) in each sample was normalized using a loading control (IgG, blue), and the fold-change of transferrin relative to the first lane was calculated. The calculated results agree closely with the predicted amount of transferrin, as shown in the Table, demonstrating the quantitative quality of the data obtained from fluorescent Western blots.
Modern improvements in digital imaging make it possible to obtain more quantitative and more reproducible data from Western blots. The availability of comprehensive imaging and analysis solutions like the FluorChem Q enables laboratories to acquire data from chemiluminescent and fluorescent blots, as well as from more routine DNA and protein gels.