Western blotting has many uses by enabling researchers to look at a single protein or subset of proteins on complex 2D gels with very high sensitivity via antibody binding. For example, Western blotting may be used to follow protein phosphorylation. An antibody that binds to all the isoforms of a poly-phosphorylated protein will show a ‘beads-on-a-string” look on a 2D gel. Western blotting is also useful for identifying known and unknown proteins in complexes brought down by co-immunoprecipitation. Once proteins of interest are located on a Western blot, 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, Jon Johansen, and Western Blot Manager, Matt Hoelter, have considerable experience Western blotting with a variety of proteins and antibodies. While the success of this method depends mostly on antibody specificity, Jon and Matt optimize conditions 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 for each Western blot.
Special WB packages using in house antibodies are now available: PhosphoTyrosine Western Blotting and PhosphoSerine/Threonine Western Blotting. Otherwise the client must supply the antibody.
Pricing and package details are available in our catalog or call 800-462-3417/email 2d@kendricklabs.com for more information.