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Calcium Binding by Ro 60 Multiple Antigenic Peptides on PVDF Membrane

Part of the Methods in Molecular Biology book series (MIMB, volume 1314)

Abstract

Antibodies directed against ribonucleoprotein (RNP) particles are observed in systemic lupus erythematosus. Ro RNP particle is one such target. It is composed of a 60 kDa protein (Ro 60 or SS-A) that is non-covalently associated with at least one of the four short uridine-rich RNAs (the hY RNAs). Previously, we showed that multiple antigenic peptides (MAPs) made from the sequence of the Ro 60 autoantigen could be used, using double-immunodiffusion studies, enzyme-linked immunosorbant assay, affinity chromatography, and surface plasmon resonance, to show intramolecular and intermolecular protein–protein interaction within the Ro 60 RNP particle. We also observed that calcium is important in mediating this interaction. We hypothesized, therefore, that 60 kDa Ro is a calcium-binding protein. To investigate this, we electrophoresed 60 kDa Ro MAPs, transferred them to PVDF membrane, and assayed calcium binding using the Quin-2 system. Several Ro 60 MAPs were found to bind calcium using this assay, as well as bovine serum albumin, another calcium-binding protein. However, a MAP constructed from the Sm autoantigen did not bind to calcium. These data, along with our observation regarding the involvement of calcium in protein–protein interaction occurring between Ro 60 antigen and Ro 60 MAPs, makes us propose that Ro 60 antigen is a calcium-binding protein.

Key words

Quin-2 Calcium binding Multiple antigenic peptides Ro ribonucleoprotein Systemic lupus erythematosus 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of MedicineUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  2. 2.Arthritis and Clinical Immunology ProgramOklahoma Medical Research FoundationOklahoma CityUSA
  3. 3.Department of Veterans Affairs Medical CenterOklahoma CityUSA
  4. 4.Oklahoma Medical Research FoundationOklahoma CityUSA
  5. 5.Helmholtz-Zentrum Dresden-Rossendorf e.V.Institute of Radiopharmaceutical Cancer ResearchDresdenGermany
  6. 6.Department of RadioimmunologyInstitute of Radiopharmaceutical Cancer ResearchDresdenGermany

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