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ODYSSEY® Fc IMAGER
Applications for the
Odyssey Fc Imaging System
MULTIPLEX WESTERNS
Application Overview
Multiplex Westerns
[ABOVE] Lysates of EGF-treated A431 cells were separated and transferred to nitrocellulose. The blot was probed with anti-ERK and anti-phospho-ERK primary antibodies, and then detected with IRDye 680LT and IRDye 800CW secondary antibodies. Blot was imaged with Odyssey Fc System for 2 min. This phospho-ERK antibody crossreacts with phospho-EGFR (upper green band).
The two infrared fluorescent detection channels of the Odyssey® Fc System enable simultaneous two-color analysis – an advantage that is not available with chemiluminescent or radioactive methods. Two primary antibodies are used, each from a different host species (e.g., rabbit and mouse). IRDye® secondary antibody conjugates (e.g., anti-rabbit and anti-mouse) are then used to detect the primary antibodies and visualize the targets.
Multiplex analysis makes normalization easy, and eliminates error introduced by stripping and reprobing or by comparison of separate blots. Superior image clarity and detail make it easier to detect subtle mobility shifts caused by protein modifications such as phosphorylation.
The Odyssey Fc System can detect Western blots in three ways:
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IR fluorescence at 700 nm
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IR fluorescence at 800 nm
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Chemiluminescence
Membrane Autofluorescence and Multiplexing
Autofluorescent background can greatly influence detection sensitivity. Low background increases the signal-to-noise ratio, making faint signals detectable and extending the linear dynamic range. At the IR wavelengths used by the Odyssey Fc System, intrinsic membrane autofluorescence is extremely low. At traditional visible wavelengths, membrane autofluorescence is much higher and can mask weaker signals.
True multiplex Western blot detection requires excellent sensitivity in both fluorescent channels – and only IR fluorescence can achieve this sensitivity. Visible fluorescence typically provides acceptable detection sensitivity in only one fluorescent channel.
[ABOVE] Nitrocellulose and PVDF membranes were imaged at 700 and 800 nm wavelengths, then imaged at 532 and 635 nm (Cy3 and Cy5 channels) with a GenePix® 4100A (Molecular Devices). Membrane background was dramatically reduced at IR wavelengths.
