How is Protein Electrophoresis Different from Western Blotting?
What is the difference between Protein Electrophoresis and Western Blotting?
The Protein Man Says:
Protein electrophoresis and Western blotting are both methods used to identify specific proteins in a sample or solution. These techniques can help researchers identify species distinctions in two plant or animal cell samples, or they may contribute to the protein identification stage of a larger study.
During protein electrophoresis, protein samples are dissolved in a solution, which may involve crushing, agitation or unfolding. The protein solution is then loaded into a well of a vertical gel matrix that is submerged in a buffer solution that conducts electricity. When positive and negative charges are applied to either end of the gel tray, the protein molecules are pushed through the gel toward the anode at the positive end. The smaller proteins move more quickly through the gel, while the larger proteins move more slowly. By the end of the process, the protein molecules have been distributed across the gel matrix according to their relative sizes. In addition to separation by relative size, or molecular weight, proteins can be separated by charge (isoelectric point) and pH.
Western blotting takes protein identification one step further; once the proteins have been distributed by electrophoresis, they can be further recognized by exposure to protein specific antibodies. Thousands of antibodies have now been isolated that react only to specific individual proteins, so these known antibodies can be applied to protein samples that have, at this stage, been identified only by molecular weight.
To remove the fractionated proteins from the gel matrix without losing their resolution, the proteins can be separated by protein electrophoresis and then transferred to a protein binding membrane. When an electrical current touches the gel matrix, the proteins are forced from the gel and onto the filter in an exact replica of their gel distribution pattern. Once the proteins are attached to the protein binding membrane, the membrane can be exposed to specific antibodies to determine which ones attach and which ones wash away. The unknown proteins can be identified by their response to specific antibodies.