Western blotting (immunostaining) has many uses because it enables researchers to focus on single proteins in a complex mixture with high sensitivity. It is especially good for identification of proteins with post-translational modifications, for example tyrosine-phosphorylation to find Src substrates. Western blotting is also an excellent way to identify unknown proteins in complexes brought down by co-immunoprecipitation. Once proteins of interest are located on the blot pattern, corresponding spots can be cut from a duplicate Coomassie blue-stained gel and identified by mass spectrometry.
Method Details: After 2D gel electrophoresis, proteins are transferred from the acrylamide slab gel to the surface of a membrane called polyvinylidene difluoride (PVDF). The PVDF is stained with Coomassie blue to show the pattern, scanned to record the image, “blocked” with a nonreactive protein such as casein to cover nonspecific binding sites, and finally incubated with an antibody against the protein of interest. After excess antibody is washed away, the blot is incubated with a secondary antibody against the primary one. The secondary antibody contains bound HRP (horse radish peroxidase) which reacts in turn with a fluorescing reagent, ECL. The light emitted by the ECL reveals the position of the protein of interest on an x-ray film which is superimposable with the Coomassie blue PVDF image showing the pattern. The latter image facilitates matching to a duplicate Coomassie gel for spot cutting for mass spectrometry. Our Lab Manager Matt Hoelter, and Western Blot Manager, Andrew Koll, have considerable experience Western blotting with a variety of proteins and antibodies. While the success of this method depends mostly on antibody specificity, conditions are optimized for each antibody to give a good blot image with low background. At least two film exposures, eg. 30 sec and 3 min, and sometimes more are provided per blot.
Special WB packages using in house antibodies are now available: PhosphoTyrosine Western Blotting and PhosphoSerine/Threonine Western Blotting.
Pricing and package details are available in our catalog or call 800-462-3417/email 2d@kendricklabs.com for more information.