Two-dimensional polyacrylamide gel electrophoresis of the protease SP220K, a renal cell carcinoma marker |
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Authors: | Mireille Starita-Geribaldi Sabine Thaon-Scarzello Maurice Le Blanc Emmanuel Van Obberghen Claudine Poustis-Delpont |
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Affiliation: | (1) Service de Biochimie, UFR de Medecine, avenue de Valombrose, 06107 Nice Cedex-2, France;(2) Unité 145, INSERM, UFR de Medecine, avenue de Valombrose, 06107 Nice Cedex-2, France;(3) Département de Chimie, U.F.R. de Sciences, Parc Valrose, 06108 Nice Cedex-2, France |
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Abstract: | The present study analyses, by two-dimensional polyacrylamide gel electrophoresis, the protease SP220K isolated from renal cell carcinoma. The pure molecule is separated using either immobilized pH gradient or isoelectric focusing in conventional carrier ampholyte in the first dimension. Some interactions with the acrylamide matrix in isoelectric focusing are discussed. The results demonstrate that two-dimensional gel electrophoresis performed with enriched media such as basolateral membranes, allows the detection of the protease. In addition, the non detection of the molecule up to now by this methodology can be explained by the high tendency of oligomerization of SP220K. Effectively the high molecular weight form of the molecule of 220 kDa is favoured in two-dimensional gel electrophoresis over monomeric forms which are better detected in SDS PAGE. This was confirmed by immunostaining performed with an antiserum to SP220K produced by nitrocellulose-bound antigen. |
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Keywords: | characterization protease renal cell carcinoma two-dimensional polyacrylamide gel electrophoresis |
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