Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract.
A two-dimensional (2-D) gel electrophoresis system in which native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) are performed subsequently to analyze protein mixtures is described. Reasonably good resolution and excellent reproducibility was obtained when the proteins in the soluble protein extract from E. coli cells were separated using this procedure. Perhaps more importantly, the relevance of this native/SDS-2-D PAGE for the detection of protein interactions in a complicated protein mixture was examined using the interaction between interleukin-2 (IL-2) and its receptor alpha chain (IL-2Ralpha) in the E. coli protein extract as a model system. Native gel was used to preserve the interactions between the two molecules and SDS gel was used to maximize the separation of the denatured proteins. Mobility changes of these two proteins on 2-D maps resulted from the formation of IL-2/IL-2-2Ralpha complex were clearly observed despite of the presence of a large number of other protein spots. Thus, this approach is a useful complement to the standard 2-D gel electrophoresis system for analyzing complicated protein mixture, especially for the study of protein interactions.
Department of Pharmaceutical and Analytical Research and Development, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA.
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D002457:Cell Extracts / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007376:Interleukin-2 / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D015375:Receptors, Interleukin-2 / Q000737:chemistry / Q000378:metabolism
D002457:Cell Extracts; D007376:Interleukin-2; D011506:Proteins; D015375:Receptors, Interleukin-2