Lab Protocols

Introduction     After proteins have been separated by electrophoresis, individual protein bands can often be identified by using an antibody that is specific to that protein.  However, to be accessible to antibodies the proteins in the gel must first be transferred from the gel to a membrane.  This process is called "Western blotting" (after E. M. Southern). We will use the "semi-dry" method of Western blotting to transfer proteins from our gels to a nitrocellulose membrane.  The semi-dry method can be used to rapidly transfer up to six gels at a time, and it does not require a large power source.  In the original description, different buffers were used for the anode and cathode, but recently it has been demonstrated that it is just as effective to use a slightly diluted version of the Tris/glycine running buffer used for SDS-polyacrylamide gels.   Materials (for one transfer) Power supply capable of 12 - 100 V, 5 W 1 -...

10X Transfer Buffer 30.3g Tris 144.1g Glycine water to 1L 1X from stock 700 mls water 100 mls 10x 200mls methanol makes 1L

The instrument Lumi-Imager was developed for the detection & accurate quantification of chemilumines-cent signals on blots & in microtiter plates. It contains a cooled CCD camera, a specially developed lens system & software. In addition to luminescence, Lumi-Imager F1 can also detect & quantify UV -excited fluorescent signals in gels. This manual describes the components & features of this instrument & some of its applications.

Direct infrared fluorescence detection on the Odyssey Infrared Imaging System provides the established standard for Western blot analysis that can’t be equaled with chemiluminescence and visible fluorescence. Infrared detection gives you the quantitative analysis and wide linear dynamic range that chemiluminescence cannot. Strong and weak bands on the same blot are accurately detected without the uncertainty and inconvenience of multiple exposures, and without spending time in the darkroom. The Odyssey System gives you clear, sharp, reproducible bands without fuzziness or “blowout”. Bands hidden by overexposure with chemiluminescence are clear in Odyssey images. Web site: http://www.licor.com/bio/odyssey/index.jsp