Western blot analysis can detect a specific protein in a mixture of any number of proteins while giving you information about the size of the protein. It is able to do this based on the use of a specific monoclonal antibody or polyclonal antibody. It does not matter whether the protein has been synthesized in vitro or extracted from cells in vivo or ex vivo. This method is, however, dependent on the use of a high-quality antibody directed against a desired protein. Thus, you must be able to produce at least a small portion of the protein from a cloned DNA fragment or purified protein which is then injected into a mouse or rabbit to generate a monoclonal antibody or a polyclonal antibody respectively. You will use this antibody as a probe to detect the protein of interest.
Western blotting tells you how much protein has accumulated in cells. If you are interested in the rate of synthesis of a protein, Radio-immunoprecipitation (RIP) may be the best assay for you. Also, if a protein is degraded quickly, Western blotting won't detect it well; you'll need to use (RIP). See the section on RIP for more information, as well as a helpful comparative chart that illustrates the differences between these two techniques.
Add 20 ul of 1X sample buffer to 20 ul of sample (or load equal concentration of protein).
Boil samples on heater for 5 - 6 minutes at 90-100 C.
Centrifuge samples after boiling. (this spins down any condensation)
Loading Samples for SDS-PAGE | Western BLot
Pour running buffer into the middle of the running apparatus.
Fill ONLY running buffer upto the bottom of the glass plates. (this prevents current leakage from the apparatus from cracks and defects)
Load Protein marker (also called protein ladder) (in different lanes when running several gels to differentiate different gels from each other)
Running the SDS-PAGE Gel for Western Blot
Run gel at 100V to 110 V for quick running OR for better resolution run gel at 35 - 40 mA (Constant current) in order to get sharp bands. This takes 2 - 2 1/2 hours to run.
Bubbles should be seen if the apparatus is working well.
Check in 5-10 minutes for migration of protein ladder downwards.
References
↑ Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, (1970)
↑
Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685, (1970)
