Western blot detection of proteins requires separation of protein mixtures by electrophoresis, followed by transfer of the separated proteins to nitrocellulose or PVDF membranes for detection. In-Gel Western detection avoids transfer problems by directly detecting target proteins within the polyacrylamide gel matrix, using the Odyssey® CLx or Classic Infrared Imaging System or the Odyssey Fc Imaging System.
The Odyssey Infrared Imaging systems allow you to detect target proteins while still embedded in the gel – without transfer to a membrane – using near-infrared secondary antibodies, such as the LI-COR® IRDye Conjugates. Using near-infrared fluorescence detection methods for In-Gel Westerns makes this a powerful technique. It saves time, reduces cost, and eliminates the variables introduced by the transfer step or subsequent blocking of the membrane. In-Gel Western detection can be performed with standard Odyssey reagents – no special kit is required.Figure 1. Sensitivity of Odyssey infrared In-Gel Westerns is equal to or better than chemiluminescence. Beginning with 10 ng/lane (far left), two-fold serial dilutions of purified Transferrin were separated by electrophoresis on duplicate gels. In-Gel Westerns were detected with infrared fluorescence (top) and chemiluminescence on film (bottom). Odyssey detection outperformed chemiluminescence.