Protein Electrophoresis (Western Blotting) Technical Handbook

Electrophoresis is a simple, rapid, and highly sensitive analytical technique to study the properties of proteins and nucleic acids, and has become a principle tool in analytical chemistry, biochemistry, and molecular biology. Electrophoresis is a particularly useful way to separate proteins prior to downstream detection or analysis (e.g. Western blot) and is a critical step in most workflows that isolate, identify, and characterize proteins.

The biological molecule of interest is contained in a sample that is generally run in a support matrix such as polyacrylamide gel or agarose. Polyacrylamide gel is widely employed to separate proteins, whereas agarose is mainly used to separate larger macromolecules such as nucleic acids. Polyacrylamide gel electrophoresis (PAGE) can be used to analyse the size, amount, purity, and isoelectric point of polypeptides and proteins.

One dimensional PAGE is a relatively simple and affordable technique. Sodium dodecyl sulfate polyacrylamide discontinuous gel electrophoresis (SDS PAGE), originally described by Laemmli, is the most commonly used system whereby proteins become separated strictly by their size, but there are different variations of this technique. However, the SDS PAGE procedure denatures / dissociates proteins and so cannot be used to analyse native proteins and those proteins whose biological activity needs to be retained for subsequent functional testing. On these occasions, it would be necessary to use a nondenaturing / nondissociating system.

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