Identification of unknown protein complex members by radiolocalization and analysis of low-abundance complexes resolved using native polyacrylamide gel electrophoresis.
Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false-positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.
Bose
Mahuya
M
Department of Physiology and Functional Genomics, College of Medicine, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, USA.
Adams
Brian P
BP
Whittal
Randy M
RM
Bose
Himangshu S
HS
D000311:Adrenal Glands / Q000737:chemistry; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D026901:Membrane Transport Proteins / Q000032:analysis*; D008928:Mitochondria / Q000378:metabolism*; D011485:Protein Binding; D012756:Sheep / Q000378:metabolism; D013462:Sulfur Radioisotopes; D053719:Tandem Mass Spectrometry
D026901:Membrane Transport Proteins; D013462:Sulfur Radioisotopes