Source Yukiko Misawa et al (University of Virginia)
Introduction
This protocol describes nuclear and cytoplasmic fractionation of tissue culture cells and a method for Western blot detection of proteins using the Odyssey Infrared Imaging System. This protocol was used to detect expression of the "small" Tap protein in 293T, HeLa and COS cells. The Odyssey system has several advantages over the more widely used chemiluminescent detection methods: 1) both Alexa Fluor 680 and IRDye800 are stable and the membranes can be stored before scanning 2) in general this system allows better quantification 3) the two-color detection system makes it possible to simultaneously detect two different proteins using secondary antibodies from different species (see Figure 1) 4) electronic data files allow the intensity of bands to be varied as needed.