High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.
Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.
Wittig
Ilka
I
Zentrum der Biologischen Chemie, Molekulare Bioenergetik, Centre of Excellence Macromolecular Complexes, Johann Wolfgang Goethe-Universität Frankfurt, D-60590 Frankfurt am Main, Germany.
Karas
Michael
M
Schägger
Hermann
H
D002021:Buffers; D004396:Coloring Agents; D003902:Detergents; D042967:Electron Transport Complex I / Q000032:analysis*; D042963:Electron Transport Complex II / Q000032:analysis*; D014450:Electron Transport Complex III / Q000032:analysis*; D003576:Electron Transport Complex IV / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008823:Micelles; D024101:Mitochondrial Proteins / Q000032:analysis*; D012394:Rosaniline Dyes
D002021:Buffers; D004396:Coloring Agents; D003902:Detergents; D008823:Micelles; D024101:Mitochondrial Proteins; D012394:Rosaniline Dyes; D042963:Electron Transport Complex II; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III; C004692:coomassie Brilliant Blue