An experimental protocol for the fractionation and 2DE separation of HeLa and A-253 cell lysates suitable for the identification of the individual antigenic proteome in Sjögren's syndrome

Abstract

Sjögren's syndrome (SS) is an autoimmune disease affecting exocrine glands, especially the salivary and lacrimal glands. Although most of the SS patients' sera have autoantibodies that can target a variety of antigens, it is not clear what determines which proteins will become autoantigens. The muscarinic receptor M3, an integral plasma membrane protein, has been proposed as a possible autoantigen in SS, and is endogenous in HeLa cells. The aim of this study was to develop a method that is able to separate and identify antigens recognised by sera from SS patients using lysates of HeLa and A-253 cells in 2D Western Blot (2DWB). The HeLa and A-253 cell lysates were fractionated in soluble and membrane-bound proteins, and the membrane-bound proteins were enriched for integral proteins. The fractions were tested using WB, confirming the presence of the main cell compartments. The rehydration solution containing ASB-14 performed better than the others in all three steps (active rehydration, focus and transfer), and efficiently separated the muscarinic receptor M3. The M3 receptor was also detected in lysates from A-253 cells. The presence of this receptor in this cell line has not been proven earlier. This work develops a suitable protocol to perform a mapping of the autoantibodies present in the sera of single SS patients, using lysates from epithelial cell lines that represent the main cell compartments as an antigen source. It is our future aim to use this protocol to perform a mapping of the antibodies present in the sera of individual SS patients.

Keywords::

• Autoantigens
• Sjögren's syndrome
• 2D
• M3 receptor
• IPG

Acknowledgements

Gustavo de Sousa has critically read the manuscript. Marianne Eidsheim and Dagny Ann Sandnes contributed valuable technical help. The confocal microscopy studies were performed at the Molecular Imaging Center (FUGE, Norwegian Research Council), University of Bergen.

Declaration of Interest: E. S. N. was supported by an EU Marie Curie Actions scholarship (MEST-CT-2005-51448). The authors thank the Broegelmann Foundation, the University of Bergen, the Meltzer Foundation and the Western Regional Health Authority for economical support. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.