Skip to main content
SpringerLink
Log in

Two-dimensional polyacrylamide gel electrophoresis of the protease SP220K, a renal cell carcinoma marker

  • Published:
Bioseparation

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adessi C, Miege C, Albrieux C & Rabilloud T (1997) Two-dimensional electrophoresis of membrane proteins: A current challenge for immobilized pH gradients. Electrophoresis 18: 127-135.

    Google Scholar 

  • Blum H, Beier H & Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93-99.

    Google Scholar 

  • Corthals GL, Molloy MP, Herbert BR, Williams KL & Gooley AA (1997) Prefractionation of protein samples prior to two-dimensional electrophoresis. Electrophoresis 18: 317-323.

    Google Scholar 

  • Diano M, Le Bivic A & Hirn M (1987) A method for the production of highly specific polyclonal antibodies. Anal. Biochem. 166: 224-229.

    Google Scholar 

  • Diano M, Le Bivic A & Hirn M (1992) Raising polyclonal antibodies using nitrocellulose-bound antigen. In: M. Manson (ed.) Methods in Molecular Biology. Vol. 10 (pp 13-22), The Humana press, Inc., Totowa

    Google Scholar 

  • Doelman CJA, Siebelder CWM & Penders TJ (1997) Routine serum protein analysis using capillary electrophoresis. Eur. J. Clin. Chem. Clin. Biochem. 35: 393-394.

    Google Scholar 

  • Endler AT, Young DS, Wold LE, Lieber MM & Currie RM (1986) Two-dimensional electrophoresis of proteins in tumours of the lung. J.Clin. Chem. Clin. Biochem. 24: 981-992.

    Google Scholar 

  • Franzén B, Iwabuchi B, Kato H, Lindholm J & Auer G (1991) Two-dimensional polyacrylamide gel electrophoresis of human lung cancer: Qualitative aspects of tissue preparation in relation to histopathology. Electrophoresis 12: 509-515.

    Google Scholar 

  • Görg A, Obermaier C, Boguth G, Csordas A, Diaz JJ & Madjar JJ (1997) Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins. Electrophoresis 18: 328-337.

    Google Scholar 

  • Görg A, Postel W, Friedrich C, Strahler JR & Hanash SM (1991) Temperature dependent spot position variability in two-dimensional polypeptides patterns. Electrophoresis 12: 653-658.

    Google Scholar 

  • Görg A, Postel W, Weser J, Günther S, Strahler JR, Hanash SM & Somerlot L (1987) Elimination of point streaking on silver staining two-dimensional gels by addition of iodoacetamide to the equilibration buffer. Electrophoresis 8: 122-124.

    Google Scholar 

  • Hochstrasser DF, Harrington MG, Hochstrasser AC, Miller MJ & Merril CR (1988) Methods of increasing the resolution of two-dimensional protein electrophoresis. Anal. Biochem. 173: 424-435.

    Google Scholar 

  • Laemmli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

    Google Scholar 

  • Lognonné JL (1994) 2D-PAGE analysis: a practical guide to principle critical parameters. Cell. Mol. Biol. 1: 41-55.

    Google Scholar 

  • Moll R, Krepler R & Franke WW (1983) Complex cytokeratin polypeptide patterns observed in certain human carcinomas. Differentiation 23: 256-269.

    Google Scholar 

  • Nawata S, Murakami A, Hirabayashi K, Sakagucchi Y, Ogata H, Suminami Y, Numa F, Nakamura K & Kato H (1999) Identification of squamous cell carcinoma antigen-2 in tumor tissue by two-dimensional electrophoresis. Electrophoresis 20: 614-617.

    Google Scholar 

  • Poustis-Delpont C, Descomps R, Auberger P, Delque-Bayer P, Sudaka P & Rossi B (1992) Characterization and purification of a guanidinobenzoatase: a possible marker of human renal carcinoma. Cancer Res. 52: 3622-3628.

    Google Scholar 

  • Poustis-Delpont C, Thaon S, Auberger P, Gerardi-Laffin C, Sudaka P & Rossi B (1994) Monomeric 55 kDa guanidinobenzoatase switches to a serine protease activity upon tetramerization. J. Biol. Chem. 269: 14666-14671.

    Google Scholar 

  • Rabilloud T, Valette C & Lawrence JJ (1994) Sample application by in-gel rehydratation improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 15: 1552-1558.

    Google Scholar 

  • Rabilloud T, Adessi C, Giraudel A & Lunardi J (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18: 307-316.

    Google Scholar 

  • Sanchez JC, Rouge V, Pisteur M, Ravier F, Tonella L, Moosmayer M, Wilkins MR & Hochstrasser DF (1997) Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 18: 324-327.

    Google Scholar 

  • Sarto C, Marocchi A, Sanchez JC, Giannone D, Frutiger S, Golaz O, Wilkins MR, Doro G, Capellano F, Hughes G, Hochstrasser DF & Mocarelli P (1997) Renal cell carcinoma and normal kidney protein expression. Electrophoresis 18: 599-604.

    Google Scholar 

  • Sheikh MI, Kagh-Hansen U, Jorgensen KE & Roigaard-Petersen H (1982) An efficient method for the isolation and separation of basolateral-membrane and luminal-membrane vesicles from rabbit kidney cortex. Biochem. J 208: 276-282.

    Google Scholar 

  • Sherwood ER, Berg LA, Mitchell NJ, Mc Neal JE, Kozlowski JM & Lee C (1990) Differential cytokeratin expression in normal hyperplastic and malignant epithelial cells from human prostate. J. Urol. 143: 167-171.

    Google Scholar 

  • Steven FS, Griffin MM & Al-Ahmad RK (1985) The design of fluorescent probes which bind to the active center of guanidinobenzoatase. Eur. J. Biochem. 149: 35-40.

    Google Scholar 

  • Svoboda M, Meuris S, Robyn C, Christophe J (1985) Rapid electrotransfer of proteins from polyacrylamide gel to nitrocellulose membrane using surface-conductive glass as anode. Anal. Biochem. 151: 16-23.

    Google Scholar 

  • Thaon S, Ferrero C, Auberger P, Michiels JF, Droz D, Amiel J, Rossi B & Poustis-Delpont C (1997) Differential SP220K expression in renal cell carcinoma and oncocytoma cells. Int. J. Cancer 72: 752-757.

    Google Scholar 

  • Thaon S, Auberger P, Rossi B & Poustis-Delpont C, (1998) SP220K is a novel matrix serine proteinase. Int. J. Cancer 77: 264-270.

    Google Scholar 

  • Tracy RP, Wold LE, Currie RM & Young, DS (1982) Patterns of normal colon mucosa and colon adenocarcinoma compared by two-dimensional gel electrophoresis. Clin. Chem. 28: 915-919.

    Google Scholar 

  • Wirth PJ, Egilsson V, Gudnason V, Ingvarsson S & Thorgeirsson SS (1987) Specific polypeptides differences in normal versus malignant human breast tissues by two-dimensional electrophoresis. Breast Cancer Res. Treat. 10: 177-189.

    Google Scholar 

  • Weiss W, Huber G, Engel KH, Pethran A, Dunn MJ, Gooley AA & Görg A (1997) Identification and characterization of wheat grain albumin/globulin allergens. Electrophoresis 18: 826-833.

    Google Scholar 

  • Westermeier R (1993) Electrophoresis in practice, VCH, Weinheim.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Service de Biochimie, UFR de Medecine, avenue de Valombrose, 06107, Nice Cedex-2, France

    Mireille Starita-Geribaldi

  2. Unité 145, INSERM, UFR de Medecine, avenue de Valombrose, 06107, Nice Cedex-2, France

    Sabine Thaon-Scarzello, Emmanuel Van Obberghen & Claudine Poustis-Delpont

  3. Département de Chimie, U.F.R. de Sciences, Parc Valrose, 06108, Nice Cedex-2, France

    Maurice Le Blanc

Authors
  1. Mireille Starita-Geribaldi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sabine Thaon-Scarzello
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maurice Le Blanc
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Emmanuel Van Obberghen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Claudine Poustis-Delpont
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Starita-Geribaldi, M., Thaon-Scarzello, S., Le Blanc, M. et al. Two-dimensional polyacrylamide gel electrophoresis of the protease SP220K, a renal cell carcinoma marker. Bioseparation 9, 133–144 (2000). https://doi.org/10.1023/A:1008198521231

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1008198521231

Search

Navigation