title: Native SDS-PAGE: high resolution electrophoretic separation of proteins with retention of native properties including bound metal ions.
abstract: Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is commonly used to obtain high resolution separation of complex mixtures of proteins. The method initially denatures the proteins that will undergo electrophoresis. Although covalent structural features of resolved proteins can be determined with SDS-PAGE, functional properties are destroyed, including the presence of non-covalently bound metal ions. To address this shortcoming, blue-native (BN)-PAGE has been introduced. This method retains functional properties but at the cost of protein resolving power. To address the need for a high resolution PAGE method that results in the separation of native proteins, experiments tested the impact of changing the conditions of SDS-PAGE on the quality of protein separation and retention of functional properties. Removal of SDS and EDTA from the sample buffer together with omission of a heating step had no effect on the results of PAGE. Reduction of SDS in the running buffer from 0.1% to 0.0375% together with deletion of EDTA also made little impact on the quality of the electrophoretograms of fractions of pig kidney (LLC-PK1) cell proteome in comparison with that achieved with the SDS-PAGE method. The modified conditions were called native (N)SDS-PAGE. Retention of Zn(2+) bound in proteomic samples increased from 26 to 98% upon shifting from standard to modified conditions. Moreover, seven of nine model enzymes, including four Zn(2+) proteins that were subjected to NSDS-PAGE retained activity. All nine were active in BN-PAGE, whereas all underwent denaturation during SDS-PAGE. Metal retention after electrophoresis was additionally confirmed using laser ablation-inductively coupled plasma-mass spectrometry and in-gel Zn-protein staining using the fluorophore TSQ.
authors.affiliation: Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N Cramer Street, Milwaukee, WI 53201, USA. petering@uwm.edu.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Nowakowski
authors.forename: William J
authors.initials: WJ
authors.lastname: Wobig
authors.forename: David H
authors.initials: DH
authors.lastname: Petering
mesh_terms: D000818:Animals; D018374:LLC-PK1 Cells; D013058:Mass Spectrometry / Q000379:methods; D008667:Metalloproteins / Q000302:isolation & purification* / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D063347:Native Polyacrylamide Gel Electrophoresis; D020543:Proteome; D013552:Swine
chemical_list: D008667:Metalloproteins; D008670:Metals; D020543:Proteome