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Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples.

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      Abstract

      There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

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      Author and article information

      Affiliations
      [1 ] Physiology, Michigan State University.
      [2 ] Center for Neurodegenerative Science Van Andel Institute.
      [3 ] Pharmaceutical Sciences, Wayne State University.
      [4 ] Pharmaceutical Sciences, Wayne State University; amosz@wayne.edu.
      Journal
      J Vis Exp
      Journal of visualized experiments : JoVE
      MyJove Corporation
      1940-087X
      1940-087X
      May 05 2017
      : 123
      28518087
      10.3791/55341

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