Cuesta, Fernando de la

González-Barderas, María

Vivanco, Fernando

Zubiri, Irene

Álvarez-Llamas, Gloria

Repositorio Institucional de la Universidad de Córdoba

48 3527(Ï0,&$1Ò0(52)(%5(52Introduction Patients with chronic kidney disease in haemo-dialysis present low quality erythrocytes, showing KLJKHU ULJLGLW\:H K\SRWKHVL]H WKDWPHPEUDQHVWUXFWXUHPD\SOD\DUROHLQWKHÀH[LELOLW\RIHU\WUR-cytes. Blue Native Polyacrylamide Gel Electropho-resis (BN-PAGE) is an electrophoretic technique for high resolution separation of membrane protein complexes in the mass range of 10KDa to 10MDa.Here we describe a methodology to perform BN-PAGE of erythrocyte membrane complexes fo-llowed by a second dimension of tricine-SDS page for isolating and identifying individual proteins forming these complexes.MethodsErythrocytes were isolated from plasma after maturation, washed in 5mM Na2HPO4 pH 8, 1mM EDTA and 0.9% NaCl and lysed in 5mM Na2HPO4pH 8, 1mM EDTA and 1mM PMSF. Membrane pe-llets were frozen at -80º C in 50 mM imidazole / HCl and 10% glycerol (pH 7) or immediately analyzed. For BN-PAGE, samples were centrifuged and pe-llets were solubilized in 50 mM sodium chloride, 50 mM imidazole, 2 mM 6- aminohexanoic acid, 1mM EDTA (pH 7) and the appropriate detergent. After centrifugation, 5 µl of 5% glycerol and Coomassie G-250 in a detergent/dye ratio of 8:1  were added to the supernatant. Membrane complexes were run in 4-13% acrylamide gradient gels. The second dimen-sion was run in 10% tricine-SDS/PAGE and a silver VWDLQLQJSURWRFROZLWKPRGL¿FDWLRQVZDVXVHG IRUtricine gels [1, 2].2D Blue native SDS-PAGE analysis of multiprotein complexes of human erythrocyte membraneIrene Zubiri1, Gloria Álvarez-Llamas1, Fernando de la Cuesta1, María González-Barderas2,Fernando Vivanco1,31Department of Immunology, Fundación Jiménez Díaz,Madrid, Spain.2Department of Vascular Pathophysiology. Hospital Nacional de Paraplejicos, Toledo, Spain.3 Department of Biochemistry and Molecular Biology, Universidad Complutense  MadridResultsMembrane proteins complexes solubilization was carried out by testing two different detergents (digitonin and Triton X-100) at various concentra-tions (0.1-10% and 0.5-2 %, respectively).  Solubi-lization was always performed for 1 h. 4% Digitonin UHVXOWHGWREHWKHEHVWRSWLRQLQWHUPVRI¿UVWGLPHQ-sion resolution, as it was the lowest detergent con-centration at which the highest number of distinct bands can be observed without disturbance from the detergent in excess. Prior to the second dimension, protein complexes disruption into individual pro-teins was optimized by treating the strips with SDS/  ҟPHUFDSWRHWKDQROVROXWLRQ WHVWHGDWYDULRXVFRP-SRVLWLRQV 6'6 ҟPHUFDSWRHWKDQRO12% SDS treatment for 1h prior to load the strip on the second dimension provided a higher number of protein spots detected; however, further increase of SDS amount negatively affected resolution. 1% ȕPHUFDSWRHWKDQROZDV XVHG DV LWV YDULDWLRQ GLGQRWVKRZDQ\FKDQJHV,GHQWL¿FDWLRQRIHU\WKURF\WHmembrane protein complexes isolated and analy-zed by 2D-BN-PAGE is being carried out by mass spectrometry.References>@ :LWWLJ,%UDXQ+3	6FKDXPOJJHU+%OXHQD-tive PAGE. Nature protocols 2006; 1:418–428.[2] Hermann Schägger. Tricine- SDS-PAGE. Nature protocols 2006; 1:16-22.

2D Blue native SDS-PAGE analysis of multiprotein complexex of human erythrocyte membrane

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Repositorio Institucional de la Universidad de Córdoba